Methods and devices for electrical sample preparation

ABSTRACT

Devices and methods are provided for electrically lysing cells and releasing macromolecules from the cells. A microfluidic device is provided that includes a planar channel having a thickness on a submillimeter scale, and including electrodes on its upper and lower inner surfaces. After filling the channel with a liquid, such that the channel contains cells within the liquid, a series of voltage pulses of alternating polarity are applied between the channel electrodes, where the amplitude of the voltage pulses and a pulsewidth of the voltage pulses are effective for causing irreversible electroporation of the cells. The channel is configured to possess thermal properties such that the application of the voltage produces a rapid temperature rise as a result of Joule heating for releasing the macromolecules from the electroplated cells. The channel may also include an internal filter for capturing and concentrating the cells prior to electrical processing.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/750,723, titled “METHODS AND DEVICES FOR ELECTRICAL SAMPLEPREPARATION” and filed on Jul. 25, 2013, which is a continuation-in-partof PCT Application No. PCT/CA2012/000698, titled “METHODS AND DEVICESFOR ELECTRICAL SAMPLE PREPARATION” and filed on Jul. 25, 2012, andclaims priority to U.S. Provisional Application No. 61/511,201, titled“METHODS AND DEVICES FOR ELECTRICAL SAMPLE PREPARATION” and filed on Jul25, 2011, and U.S. Provisional Application No. 61/586,906, titled“METHODS AND DEVICES FOR ELECTRICAL SAMPLE PREPARATION” and filed onJan. 16, 2012, the entire contents of which are all incorporated hereinby reference.

BACKGROUND

This disclosure relates to methods of preparation and processing ofbiological samples. More particularly, the disclosure relates to theprocessing of biological samples to be used for performing diagnosticassays and for therapeutic uses.

Despite unprecedented progress in measurement techniques over the recentyears, satisfactory noninvasive measurements of target analytes inbiological samples are still not possible in most cases. Generally, oneor more sample pretreatment steps are necessary. These steps arereferred to as sample preparation, the goal of which is to render a rawsample suitable for a measurement with a satisfactory signal to noiseratio. The sample preparation is accomplished by proceeding throughcleanup, enrichment or concentration, and medium balance steps. Inaddition, in the case of measuring cellular contents, the molecules ofinterest must be released to the medium via cell lysis. Frequently, alysate treatment step, involving various reagents, is needed to make thelysate assay-ready by modifying the target and non-target molecules oradjusting the lysate composition. Sample preparation is often thebottleneck in the measurement process, as it tends to be slow andgenerally involves multiple reagents and manual steps that requiresubstantial time, complexity and cost.

The complexity of sample preparation can be better appreciated byreferring to a typical example in which pathogenic bacteria in humanurine are identified through rRNA hybridization where a specificsequence on the 16S rRNA is hybridized with a labeled complementarynucleic acid probe. An exemplary sample preparation protocol employs theforetold five steps as follows: 1) relatively large particles, such ascrystals, and excess ions are removed (cleanup); 2) the bacteria countper unit volume is increased by reducing the water (liquid) content(enrichment); 3) the ribosomes are released by lysing the cells (lysis);4) the lysate is treated such that the rRNA is partially untangled fromthe accompanying proteins and its conformation is modified to betterexpose the target region to the probes (lysate treatment); and 5) thechemical and ionic composition of the lysate is adjusted to supporthybridization (medium balance).

In some instances, the sample preparation is further complicated by theneed to process the sample with various reagents. For example, reagentsmay be needed for lysis, binding and elution, precipitation, removal orinhibition of interferants and/or contaminants, denaturing of DNA toobtain single stranded DNA, separation of rRNA from ribosomal proteins,and denaturing of enzymes or other proteins such as DNAse and RNAse. Oneexample of a reagent treatment for the purification of nucleic acidsinvolves cell lysis and molecular denaturation by a chaotropic agent(guanidinium thiocyanate) followed by nucleic acid extraction byphenol-chloroform liquid-liquid separation, which is a complex methodinvolving very hazardous reagents. Further, sample processing byreagents may involve additional steps such as heating and bead milling,for example, to enhance the function and effectiveness of reagents.

SUMMARY

Devices and methods are provided for electrically lysing cells andreleasing macromolecules from the cells. A microfluidic device isprovided that includes a planar channel having a thickness on asubmillimeter scale, and including electrodes on its upper and lowerinner surfaces. After filling the channel with a liquid, such that thechannel contains cells within the liquid, a series of voltage pulses ofalternating polarity are applied between the channel electrodes, wherethe amplitude of the voltage pulses and a pulsewidth of the voltagepulses are effective for causing irreversible electroporation of thecells. The channel is configured to possess thermal properties such thatthe application of the voltage produces a rapid temperature rise as aresult of Joule heating for releasing the macromolecules from theelectroporated cells. The channel may also include an internal filterfor capturing and concentrating the cells prior to electrical processingand removal of cellular debris from the cell lysate after electricalprocessing.

Accordingly, in a first embodiment, there is provided a method ofelectrically processing a liquid within a microfluidic device to releasemacromolecules from at least one cell within the liquid;

-   -   the microfluidic device including:        -   an upper planar substrate formed from a thermally insulating            material;        -   a lower planar substrate formed from a thermally insulating            material; and        -   a side wall having a thickness on a submillimeter scale,            wherein said upper planar substrate, said lower planar            substrate and said side wall define a channel;        -   an upper electrode provided on an inner surface of said            upper planar substrate; and        -   a lower electrode provided on an inner surface of said lower            planar substrate;    -   the method including:        -   flowing the liquid into the channel;        -   applying bipolar voltage pulses between the upper electrode            and the lower electrode and electrically heating the liquid            to an elevated temperature between approximately 30 degrees            Celsius and 250 degrees Celsius;        -   wherein the voltage pulses are applied such that the liquid            is heated faster than a timescale of thermal diffusion from            the channel; and        -   wherein the voltage pulses are applied such that a rate of            change of the temperature of the liquid is sufficient to            effect lysis of the at least one cell.

In another aspect, there is provided a microfluidic device forprocessing a liquid to release macromolecules from at least one cellwithin the liquid, the microfluidic device comprising:

-   -   an upper planar substrate formed from a thermally insulating        material;    -   a lower planar substrate formed from a thermally insulating        material; and    -   a side wall having a thickness on a submillimeter scale, wherein        said upper planar substrate, said lower planar substrate and        said side wall define a channel;    -   an upper electrode provided on an inner surface of said upper        planar substrate; and    -   a lower electrode provided on an inner surface of said lower        planar substrate;    -   a first port in flow communication with a first side of said        channel;    -   a second port in flow communication with a second side of said        channel; and    -   a voltage controller configured to:        -   apply bipolar voltage pulses between the upper electrode and            the lower electrode and electrically heat the liquid to an            elevated temperature between approximately 30 degrees            Celsius and 250 degrees Celsius; and        -   apply the voltage pulses such that the liquid is heated            faster than a timescale of thermal diffusion from the            channel, and such that a rate of change of the temperature            of the liquid is sufficient to effect lysis of the at least            one cell.

A further understanding of the functional and advantageous aspects ofthe disclosure can be realized by reference to the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will now be described, by way of example only, withreference to the drawings, in which:

FIG. 1 shows a schematic of an electrical sample processing device.

FIG. 2 shows a schematic cross-sectional view parallel to the flow ofthe electrical sample processing device.

FIG. 3 shows schematic cross-sectional views of the electrical sampleprocessing device, which has been equipped with sample cleanup andsample concentration capability, where (a) shows a filter dividing thechannel into two portions, and where (b) illustrates the use ofmicrospheres as an example material for supporting a filter within thechannel.

FIG. 4(A) shows a schematic cross-sectional view of the electricalsample processing device which has been equipped with an alternativesample cleanup and sample concentration capability.

FIG. 4(B) shows a schematic plan view of the electrical sampleprocessing device of FIG. 4 a.

FIG. 4(C) provides a flow chart illustrating a method of electricalprocessing of cells within a liquid.

FIG. 5(A) shows an equivalent circuit model for the device of FIG. 2.

FIG. 5(B) shows an approximation to the equivalent circuit in FIG. 5 a.

FIG. 5(C) shows the current response of two typical channels using SEOA1or SEOA2 electrodes to a bipolar pulse.

FIGS. 5(D) and 5(E) schematically depict the plan FIG. 5(D) and crosssectional

FIG. 5(E) views of an example electrical chamber.

FIG. 5(F) is an example implementation of a voltage controller.

FIG. 5(G) shows the block diagram of the impedance based temperatureregulation scheme.

FIG. 6 shows the measured current envelope for an open channel.

FIG. 7 shows the channel temperature distribution for the open channeljoule heating analysis.

FIG. 8 shows the temporal dynamics of the maximum channel transienttemperature joule heating analysis.

FIG. 9 shows the time dependent peak current envelope for channel with ashutoff valve at inlet and outlet ports in an open and closed position.

FIG. 10 shows a schematic cross-sectional view of the electrical sampleprocessing device, which has been equipped with a pressure regulationcapability.

FIG. 11 shows the sample pre-treatment module for reducing ioniccontent.

FIG. 12(A) shows the measured current envelope for the channel in whichthe enzyme suspensions were subjected to electrical treatment.

FIG. 12(B) shows the measured residual enzyme activity following theelectrical treatment with different test parameters.

FIG. 13 illustrates the dose response curve of Bradford protein assaydetermined by assaying BSA standards.

FIG. (A) shows the measured current envelope for the channel in which E.Coli suspension underwent electrical lysis under different test

FIG. 14(B) shows the effects of pulse amplitude on the electrical lysisperformance of the device as determined by quantifying the release ofproteins from E. coli cells.

FIG. 14(C) shows the effects of pulse amplitude on the electrical lysisperformance of the device as determined by quantifying the release ofnucleic acids from E. coli cells.

FIG. 14 (D) shows the effects of pulse amplitude on the electrical lysisperformance of the device as determined by measuring the hybridizationof 16 rRNA released from E. coli cells to specific probes.

FIG. 15(A) shows the measured current envelope for the channel in whichE. Coli suspension underwent electrical lysis under different testparameters and constant pulse amplitude.

FIG. 15(B) shows the effects of train duration and ionic strength on theelectrical lysis performance of the device as determined by quantifyingthe release of proteins from E. coli cells.

FIG. 15(C) shows the effects of train duration and ionic strength on theelectrical lysis performance of the device as determined by quantifyingthe release of nucleic acids from E. coli cells.

FIG. 15(D) shows the effects of train duration and ionic strength on theelectrical lysis performance of the device as determined by measuringthe hybridization of 16S rRNA released from E. coli cells to specificprobes.

FIG. 15(E) shows the effects of train duration and ionic strength on theactivity of E. coli cells-associated beta-galactosidase after electricallysis.

FIG. 15(F) shows the release of nucleic acids from E. coli cells byelectrical lysis, visualized after resolving total nucleic acids onagarose gel electrophoresis.

FIG. 15(G) illustrates a downstream application of genomic DNA in the E.coli cells cell lysate prepared by electrical lysis. The supernatant ofcell lysate was subjected to PCR amplification of the bacterial 16S rDNAgene fragment. The PCR product was visualized after resolving on agarosegel electrophoresis.

FIG. 16(A) shows the measured current envelopes for the channel in whichthe purified plasmid DNA and E. coli cells GB lysate for FOR wereelectrically treated.

FIGS. 16(B) and 16(C) illustrate conformational change in nucleic acidsas a result of electrical treatment of purified NH5-α genomic DNA andpurified pUC19 plasmid DNA.

FIG. 16(D) shows PCR-ready quality of cell lysate prepared by electricaltreatment. The supernatant of glass bead cell lysate was seriallydiluted or electrically treated prior to PCR amplification of thebacterial 16S rDNA gene fragment. The PCR product was visualized afterresolving on agarose gel electrophoresis.

FIG. 16(E) shows the plasmid DNA purified from the supernatant of celllysate.

FIGS. 16(F)-(I) demonstrate the integrity of plasmid DNA in thesupernatant of cell lysate by culturing E. coli on a selection mediumafter transformation with purified pUC19 plasmid, showing plated results16(F) without plasmid, 16(G) with purified plasmid control, 16(H) withpurified plasmid from glass bead lysis and 16(I) with purified plasmidfrom E-lysis.

FIG. 17(A) shows the measured current envelopes, corresponding to 4parameter sets, for the channels in which Streptococcus pneumoniae cellswere electrically lysed.

FIG. 17(B) shows the effects of voltage pulse amplitude and ionicstrength on the electrical lysis performance of the device as determinedby quantifying the release of proteins from Streptococcus pneumoniae.

FIG. 17(C) shows the effects of voltage pulse amplitude and ionicstrength on the electrical lysis performance of the device as determinedby quantifying the release of nucleic acids from Streptococcuspneumoniae.

FIGS. 17(D) and 17(E) show spectra of nucleic acids (17D) and PCRamplification of the bacterial 16S rDNA gene fragment derived fromStreptococcus pneumoniae, visualized after resolving on agarose gelelectrophoresis.

FIG. 18(A) shows the measured current envelopes, corresponding todifferent devices used for lysing Streptococcus pneumoniae cells.

FIGS. 18(B) and 18(C) show the effects of electrode material on thelysis performance of the cells as determined by quantifying the releaseof 18(B) proteins and 18(C) nucleic acids from Streptococcus pneumoniae.

FIG. 18(D) shows PCR amplification of the bacterial 16S rDNA genefragment of Streptococcus pneumoniae as visualized after resolving onagarose gel electrophoresis.

FIG. 19(A) shows the measured current envelopes for the channels inwhich S. cerevisiae cells were electrically lysed, for both restrictedand open channels.

FIG. 19(B) FIGS. 19(B) and 19(C) demonstrate lysis efficiency of theelectrical lysis method, as compared to that of glass bead lysis,according to the measured 19(B) total protein concentration and 19(C)total nucleic acid released from lysed S. cerevisiae cells.

FIG. 20(A) shows the measured current envelopes, corresponding todifferent devices used for lysing E. coli cells.

FIG. 20(B) shows reverse transcription (RT)-PCR amplification of asection of E. coli rRNA visualized after resolving on agarose gelelectrophoresis (S=supernatant, T=total lysate).

FIG. 21 shows the measured current through an aqueous media subjected toelectrical pulse trains inside electrical channels with open andrestricted inlet and outlet ports.

FIG. 22 shows the calculated time dependent average temperatures of therestricted and open channels with the current responses presented inFIG. 21.

FIG. 23 lists the electrical and corresponding thermal conditions forthe lysis of fungal cells used to demonstrate the dependence of thelysis efficiency on the electrical channel temperature.

FIG. 24(A) shows the fluorescence signal measured during real timeRT-PCR assay for different conditions in Example 6.2.

FIG. 24(B) shows the CT values of the real time RT-PCR assay describedin FIG. 24a for the detection of fungal cells lysed under differentconditions.

FIG. 25(A) shows the measured current through a cell suspensionsubjected to electrical pulse trains inside the restricted electricalchannel corresponding to the condition 1 of Example 6.3.

FIG. 25(B) shows the measured current through a cell suspensionsubjected to electrical pulse trains inside the restricted electricalchannel corresponding to the condition 2 of Example 6.3.

FIG. 25(C) shows the measured current through a cell suspensionsubjected to electrical pulse trains inside the restricted electricalchannel corresponding to the condition 3 of Example 6.3.

FIG. 25(D) shows the measured current through a cell suspensionsubjected to electrical pulse trains inside the restricted electricalchannel corresponding to the condition 4 of Example 6.3.

FIG. 25(E) shows the measured current through a cell suspensionsubjected to electrical pulse trains inside the restricted electricalchannel corresponding to the case when the pulse density was kept at themaximum value over the whole duration of the pulse train.

FIG. 26(A) shows the fluorescence signal measured during real timeRT-PCR assay for different conditions of Example 6.3.

FIG. 26(B) shows the CT values of the real time RT-PCR assay describedin FIG. 26(a) for the detection of fungal cells lysed under differentconditions.

FIG. 27(A) shows the applied voltage and measured current through a cellsuspension subjected to electrical pulse trains inside an openelectrical channel.

FIG. 27(B) shows the applied voltage and measured current through a cellsuspension subjected to electrical pulse trains inside a closedelectrical channel.

FIG. 27(C) shows the measured impedances of the open and closed channelswith electrical responses presented in FIGS. 29a and 29 b.

FIG. 28(A) qualitatively demonstrates lysing efficiency of fungal cellsin open and closed channel electrical channels by detection of RT-PCRamplified product of Candida albicans 18S rRNA fragment, as wasvisualized after resolving on agarose gel electrophoresis.

FIG. 28(B) quantitatively demonstrates lysing efficiency of fungal cellsin open and closed channel electrical channels by detection of RT-PCRamplified product of Candida albicans 18S rRNA fragment, as detected bymonitoring the hybridization of molecular beacon probes.

DETAILED DESCRIPTION

Various embodiments and aspects of the disclosure will be described withreference to details discussed below. The following description anddrawings are illustrative of the disclosure and are not to be construedas limiting the disclosure. Numerous specific details are described toprovide a thorough understanding of various embodiments of the presentdisclosure. However, in certain instances, well-known or conventionaldetails are not described in order to provide a concise discussion ofembodiments of the present disclosure. It should be understood that theorder of the steps of the methods disclosed herein is immaterial so longas the methods remain operable. Moreover, two or more steps may beconducted simultaneously or in a different order than recited hereinunless otherwise specified

As used herein, the terms, “comprises” and “comprising” are to beconstrued as being inclusive and open ended, and not exclusive.Specifically, when used in the specification and claims, the terms,“comprises” and “comprising” and variations thereof mean the specifiedfeatures, steps or components are included. These terms are not to beinterpreted to exclude the presence of other features, steps orcomponents.

As used herein, the term “exemplary” means “serving as an example,instance, or illustration,” and should not be construed as preferred oradvantageous over other configurations disclosed herein.

As used herein, the terms “about” and “approximately”, when used inconjunction with ranges of dimensions of particles, compositions ofmixtures or other physical properties or characteristics, are meant tocover slight variations that may exist in the upper and lower limits ofthe ranges of dimensions so as to not exclude embodiments where onaverage most of the dimensions are satisfied but where statisticallydimensions may exist outside this region. It is not the intention toexclude embodiments such as these from the present disclosure.

In selected embodiments disclosed below, methods and devices areprovided for subjecting a sample to electrical lysis of cells and/or theelectrical treatment of molecular species within the sample. Methodsdisclosed below involve subjecting a liquid sample to an amplitudemodulated electrical pulse train in a confined fluidic channel for thecell lysis and/or treatment of macromolecules. The pulse train generatesa pulsed electric field across the thickness of the channel andresponsively generates heat within the channel, where the thickness issmall compared to at least one other dimension of the channel. Thechannel may be closed or open during the application of the voltagepulses, as further described below.

In selected embodiments in which the sample may or may not includecells, an electric field is applied as a series of pulses wherebymacromolecules within the sample are treated or processed. Withoutintending to be limited by theory, it is believed that as the voltagepulse train acts on the liquid within the channel, appreciable ioncurrent is established. This causes the liquid medium (generally, anaqueous medium) to be rapidly heated by Joule heating, thereby providingan electrical treatment mechanism to macromolecules within the liquidmedium. The maximum temperature depends on the timescale of the electricpulse train, the applied voltage, the ionic strength of the liquid, theelectrical and thermal characteristics of the channel and the pressureregulation of the channel.

The channel temperature may be maintained at the desired temperature fora period, termed herein as the residence time, by passive feedbackthrough conductivity change arising from liquid-gas phase transition, oractive feedback through current measurements.

Due to the large surface to volume ratio of the liquid confined in thechannel, and the thermal properties of the channel (described in furtherdetail below), and the sub-second duration of the pulse train, theheating and cooling of the liquid is rapid. In the context of thepresent disclosure, this process of heating and cooling in sub-secondtime scale is referred to as “flash heating”. In some embodiments, theheating rate is faster than a timescale for thermal diffusion.

In one embodiment, in which the liquid sample provided within thechannel includes cells, the application of the electric pulses causesthe cells to undergo irreversible electroporation. The mechanism ofirreversible electroporation of microorganisms is not completelyelucidated in the literature. It has been postulated that when a pulsewith high enough intensity is applied to the cell suspension, such thata voltage higher than 1 V is established across the cell membrane, poresare generated in the membrane. The process is known as electroporation.When the number of pulses and/or pulse-width is large enough, the cellmembrane will be ruptured, permanently compromising the integrity of thecell membrane. This process is known as irreversible electroporation,which is accompanied by the release of some intracellular contents fromthe cell. In general, the electric field intensity required to achievethe irreversible electroporation of microorganisms is greater than about10 kV/cm. For example, many microorganisms can be subjected toirreversible electroporation for electric fields within the range ofapproximately 12-45 kV/cm. The action of such an intense electric fieldin the suspension medium is accompanied by appreciable ion currents,which for the device disclosed herein increases the temperature of themedium via Joule heating. This may increase the efficacy of irreversibleelectroporation, lowering the threshold of irreversible electroporationand contributing to cell membrane/wall damage as the heating has asignificant influence on cell membrane fluidity and stability.

Without wanting to be limited by theory, it is believed that the effectsof irreversible electroporation and heat induced cell disintegration acttogether to enhance the release of cellular contents. The combinedprocess by which desired macromolecules are released from cells will behenceforth referred to as “electrical lysis”.

In the case of microorganisms with protective cell walls in addition tothe cell membrane, the influence of irreversible electroporation alonemay be insufficient for releasing some of the cellular contents ofinterest when not aided by this heating. The heating may be responsiblefor disintegration of the cell wall to the extent that it enablesrelease of higher molecular weight cellular contents.

Without being bound to any theory, it is believed that the intenseelectric field may cause structural modification in macromolecules, suchas proteins, due to a modification of the balance of forces thatmaintains their native structures. The alterations in conformation maydisengage the chemically reactive functional groups on a macromoleculeand render it incapable of performing its catalytic or other designatedfunctions. However, macromolecules are generally more resistant toelectric fields than comparatively larger microorganisms. Excess heatacting for a period of up to 100 ms, can cause excessive thermalfluctuation of structure and can deprive some of the macromolecules oftheir functionality. Generally speaking, flash heating and the intenseelectric field contribute to the electrical treatment mechanismdescribed herein, which causes macromolecular species within the liquidto experience irreversible conformation changes. In selectedembodiments, the electrical treatment method described above is employedsuch that the sample (such as a lysate for example) is rendered moresuitable for downstream applications such as diagnostic assays. Theelectrical lysis of cells and subsequent electrical treatment of thelysate is, either singly or in combination, termed electrical processingthroughout this disclosure.

As further described below, the ionic strength of the sample may beselected to be below a maximal value in order to support theestablishment of an effective electric field with a suitable timescalefor effecting electrical processing. The specific maximal value or rangeof suitable values of the ionic strength will mainly depend on thecapability of the applied voltage source to deliver high voltage alongwith the corresponding current over the timescale over which theprocessing is desired to occur. It is to be understood that thoseskilled in the art may perform routine experimentation in order todetermine a suitable upper limit or range of values for the ionicstrength in a given application. Since both electrical lysis andelectrical treatment are performed according to selected embodiments ina medium with low ionic strength and in the absence of additionalreagents, a post lysis medium balance step, generally required intraditional sample preparation methods, may not be necessary. In otherapplications, the ionic strength suitable for a subsequent step can beeasily altered by adding an appropriate concentration of ions to theprocessed sample or lysate.

In some embodiments, additional sample processing steps such as samplecleanup and sample concentration may be readily incorporated into thedevice, potentially without adding significant complexity or cost to thedevice or the manufacturing process. Examples of the incorporation ofsuch additional processing steps are described in further detail below.Accordingly, selected embodiments provided herein include devices andmethods that enable the direct and rapid processing of samples withoutrequiring additional reagents and additional processing steps.

In selected embodiments, devices and methods are provided forpreparation of samples containing microorganisms, such as, but notlimited to, blood, urine or a growth medium, for diagnostic assays. Thedevice, which may be provided in the form of a disposable cartridge, canbe optionally interfaced to reaction chambers where the target moleculesundergo a detection or identification process.

Referring now to FIG. 1, an example embodiment of a device forperforming electrical sample processing is provided. FIG. 1 shows anexample configuration of the device 1, while more details are providedby the schematic cross section of shown in FIG. 2. The device has a thinchannel 2 (where the thickness of the channel is small compared tolateral dimensions of the channel) which is defined on one side by thebase plate 5, insulating layer 9 and electrode 7 and on the oppositeside by top plate 6, insulating layer 10 and electrode 8. The upper andlower portions are separated by a thin spacer, in which material isremoved to form the channel cavity. Typically, the spacer is made of adielectric material which may be slightly deformable under an appliedclamping pressure, or which is bonded to the upper and lower surfaces ofthe channel cavity. The spacer thus defines the side walls of thechannel, provides the fluid seal, and electrically insulates the top andbottom electrodes from each other.

In alternative embodiments, the device need not include a separatespacer, and side walls defining the lateral portions of the channel maybe formed, at least in part, within a substrate, such that the substrateincludes a recessed portion having a bottom surface and lateral sidewalls adapted to form at least a portion of the channel.

The lower electrode 7 and upper electrode 8 are electrically isolatedfrom the base and top plates (substrates) by lower and upperelectrically insulating layers, 9 and 10. In an alternative embodiment,one or both of the upper and lower plates are nonconductive and theelectrical insulating layer may be omitted. In accordance with thermalrequirements, thermal insulating layers may also be provided which maybe separate from or be at least a portion of the electrical insulatingmaterial.

The channel includes an inlet port 3 through which fluid sample andother fluids may be introduced and which may be in fluid communicationwith upstream chambers where pre-filtering may optionally be performedand which may include chambers where fluids are stored. The device isalso equipped with an outlet port 4 that may be in fluid communicationwith a collecting apparatus or chamber, such as a waste chamber ordownstream assay reaction chamber. Flow along the channel is provided bya pressure differential between inlet and outlet ports. The device mayinclude additional fluid features, such as valves for opening andclosing ports 3 and 4.

The channel has dimensions H×W×L which, in one example implementation,may be on the order of 0.1×5×10 mm³, but which may be greater or lesserin accordance with operational requirements. Two electrodes 7 and 8 areintended for inducing an electric field across the channel.

The microfluidic channel has a thickness on a submillimeter (i.e.micron) scale. An example range for the channel thickness is 5 μm<H<1000μm. In some embodiments, a suitable channel thickness for obtainingeffective electrical lysis and treatment with practical voltage sources(for example, in the 10-200 V range) may be between approximately 50 μmand 500 μm. The channel length and width may be selected to provide asuitable channel volume and optionally a suitable flow rate through thechannel. Without excluding the use of the methods and devices describedherein for microfluidic applications in which the channel length andwidth are also on a submillimeter scale, more typical example ranges forthe length and width of the channel are approximately 1 mm<W<10 mm and 5mm<L<50 mm.

In one example implementation, one or more of the first and secondelectrodes may be provided as metal coatings that are deposited onelectrically insulating layers provided on the upper and lower channelsurfaces. Example thicknesses for the deposited electrode include 1nm<h<1 μm, although thicker layers may also be realized.

In another example, one or more of the first and second electrodes maybe provided as metal foils, for which material properties and dimensionsare chosen in accordance with electrical and thermal requirements.Example thicknesses for the metal foil include 10 μm<h<500 μm, or 20μm<h<200 μm.

In embodiments intended for samples which contain cells or viruses,collectively referred to here as cells, the channel may be operated toconcentrate the cells prior to electrical lysis. Applying a timedependent unipolar voltage on the electrodes, an electric field isestablished in the channel, which exerts an effective force on chargedcells and carries them to a thin region at the immediate vicinity of theanodic electrode while the excess fluid is carried out of the outletport. For improved retention of the cells, the anodic electrode may becoated with capture ligands specific to a class of cells that aredesired to be retained. As the cells, concentrated at the lowerextremity of the channel, slowly move over the electrode, theyspecifically bind or hybridize with their corresponding capture ligands.This mode of sample concentration has been disclosed in co-pendingpatent PCT Application Number WO/2011/014946A1.

An alternative embodiment in which the device is adapted forconcentration and cleanup capabilities is presented in FIG. 3(A). Afilter 16, for example a membrane filter, having a thickness less thanthat of the channel (or equivalently, the channel spacer) is securedwithin the channel such that the channel is divided into two portions 14and 15, thereby enabling cells (or other particulate matter) within thesample to be retained by the filter as the sample is flowed between theinlet port 12 and outlet port 13. In some embodiments, at least aportion of the filter is placed between the upper and lower electrodes.

In one example implementation, the filter may be made of chemical and/orthermal resistant material, such as high density polyethylene, orpolycarbonate membrane. For example, a thermally resistant filter may bebeneficial in applications involving rapid thermal treatment to avoiddegradation of the filter during electrical processing. Since suchmembrane filters are typically very thin, for example, approximately10-20 microns, a support may be required to prevent the collapse of thefilter onto the channel surface due to fluid pressure, which may preventfluid flow through the device. Filter support may be added in the formof structures introduced into the channel which retain the electricalisolation of the upper and lower electrodes and which do notsignificantly impede sample flow. In one embodiment, a suitable filtersupport is monodispersed microspheres 17 which are bound to the membranefilter or the channel surface as shown in the cross-section diagram ofFIG. 3(B). In another embodiment, the filter support may be provided byadditional spacer structures located on either side of the filter, wherethe additional spacers are placed such that they do not substantiallyimpede the flow of liquid within the channel. The filter support and/orthe filter may be selected to be formed from a material havingdielectric properties suitable for inducing the electric field to flowthrough the liquid within the channel, as opposed to bypassing theliquid and flowing through the filter and filter support.

An alternative embodiment of the device adapted for concentration andcleanup capabilities is presented in cross section view A-A in FIG. 4(A)and plan view in FIG. 4(B). A filter 38, for example a membrane filteras described above, is secured within a chamber 33 fluidically connectedto the inlet port 3, the electrical channel 2 and the filter outlet port34. A diffuser support 39 is provided for the filter if necessary. Whenthe channel inlet valve 36 is closed and the inlet port valve 35 and thefilter outlet port valve 37 are open sample fluid can be flowed throughthe filter from the inlet port 3 to the filter outlet port 34 and thento a waste chamber in fluid connection with outlet port 34 and therebycells within the sample are retained by the filter. A resuspension fluidof suitable composition and ionic strength for subsequent electricalprocessing and downstream processes is then passed from the inlet port 3until the sample fluid has been sufficiently cleared through the filteroutlet port 34. The filter outlet port 37 is then closed and the channelinlet port 36 is opened and the resuspension fluid is flowed from theinlet port thereby carrying the retained cells to the electrical channelfor subsequent electrical processing. Alternatively the retained cellsare resuspended and carried to the electrical channel by flow from thefilter outlet port 34 while the filter outlet port 37 is open and theinlet port 35 is closed. Electrical contacts 41 are provided for theapplication of the electric field.

The passage of a large volume of sample fluid through the filtertogether with the relatively small volume of resuspension fluid used tocarry the retained cells to the electrical channel allows the cellconcentration for subsequent lysing and treatment to be arbitrarilyincreased from the initial sample concentration. It also allows for thesubstitution of the sample fluid by a suspension fluid appropriate fordownstream processes including electrical lysing and electricaltreatment and subsequent assays. The foregoing is one of a number ofdifferent arrangements of valves and fluid movements that produceequivalent functionality which can be varied by those skilled in the artbased on the present disclosure.

The application of a suitable amplitude modulated electric pulse trainon the two electrodes causes the cells to lyse. Depending on themagnitude and duration of the resulting electric field, the electricfield and its associated temperature rise causes or induces moleculessuch as proteins and nucleic acids to be released from the cell as alysate. The released molecules further undergo a transformation in theperiod between release and cooling down of the liquid. These processesconstitute what are identified herein as electrical lysis and electricaltreatment, which can provide useful steps in the preparation of cellularbiological samples for diagnosis or other purposes. As discussed above,the underlying mechanisms enabling these electrical processing steps arebelieved to result from the electrical and thermal response in thechannel from the application of the prescribed pulse train.

It is believed that the operation of the device involves establishing anelectric field with consequent electric current in the channel ofsufficient magnitude to cause electrical lysis of cells and/orelectrical treatment of macromolecules in the channel fluid. Forenhanced performance the electrolysis of the fluid at the electrodeinterface with its attendant gas production and bubble formation shouldbe minimized. Some embodiments provided herein accomplish this byinsulating one or more of the electrodes from the sample with a thinlayer of dielectric coating, thus forming a blocking electrode that isfree from direct electrical communication with liquid within thechannel, which serves to eliminate any charge transfer processes fromoccurring across the electrode-electrolyte interface.

In other embodiments, the channel may include non-blocking electrodesthat are capable of directly contacting the liquid, thereby supporting aFaradic current. The electrolysis products are avoided by operating thedevice using a high frequency bipolar pulse train. The ions produced atthe liquid-electrode interface are significantly neutralized inalternating cycles before significantly diffusing away into the bulkmedium. Unfortunately the presence of the Faradic current at theinterface supports redox reactions which may damage the macromoleculesin the proximity of the electrode. This effect could be alleviated byproviding a protective permeation layer. Generally speaking, thepermeation layer may be provided for allowing the movements of soluteions to the electrode while preventing the macromolecules from reachingthe electrolyte-electrode interface.

Exemplary methods for preparing a solid support with a permeation layerare henceforth described. These examples are intended to benon-limiting, and it is to be understood that any suitable material orcoating may be provided that permits solute ion transport to theelectrode while restricting macromolecule transport. Permeation layermaterials may optionally include functional groups for achieving thedesired selective transport function of the layer.

Examples of surface preparations are the deposition of small moleculessuch as organosilanes and thiol linkers by covalent interaction ormacromolecules such as poly-L-Lysine and PEI by physical adsorption.

In an exemplary, yet non-limiting embodiment, a heterobifunctionalsilane layer with functional groups, X-Si-X′, can be deposited on anysurface (Y) on which a silane layer can be applied to form Y-O-Si-X′. X′may be trimethoxy (—OCH₃)₃, triethoxy (—OC₂H₅)₃ or trichloro (Cl₃) andform Y—O—Si—X′ chemistry upon hydrolysis.

One example of such a surface is a hydroxylated surface of aluminum,with a naturally or artificially processed oxide layer, and aheterobifunctional silane layer that is generated by Al—O—Si—X′formation. X may vary and covalently interacts with the respectivefunctional group of any additional molecule to be attached to the silanelayer via any appropriate chemistry. For example, X can be glycidylfunctional group of glycidyloxipropyl-trimethoxysilane (GOPTS) or aminofunctional group of 3-aminopropyltriethoxysilane (APTS). Glycidylfunctional group of GOPTS will interact readily with an amino functionalgroup of the molecule to be attached. An additional activation of aminofunctional group of APTS with any crosslinking chemistry, for example,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) andN-hydroxysuccinimide (NHS), may be provided for covalent interactionwith a carboxyl functional group of the molecule to be attached.Optionally, amino functional group of APTS can be pre-activated with anyknown chemistry, for example, with a glutaraldehyde homobifunctionalcrosslinker, to interact readily with amino functional group of themolecule to be immobilized.

Alternatively, protein molecules with high affinity and specificity suchas avidin or streptavidin can be immobilized on the functionalizedsurface via any suitable chemistry and a biotinylated molecule can bereadily immobilized on the surface by biotin-avidin affinityinteraction.

The surface can be prepared by procedures presented in the followingnon-limiting example. Polished aluminum support plates were cleaned withwater then rinsed twice with methanol and air-dried. 2% 3-AminopropylTriethoxysilane is prepared in 95% Methanol 5% water and the plates wereimmersed in silane for 5 min. Then, the plates were rinsed in methanoltwice, air-dried and baked at 110° C. for 10 min. After cooling, theplates were immersed in 2.5% glutaraldehyde homobifunctional crosslinkerin phosphate buffered saline, pH 7.4 at room temperature for 1 hour. Theplates were rinsed thoroughly with water and air-dried.

In another embodiment the permeation layer can be a hydrogel, such as apolyacrylamide based network-like hydrogel (e.g., Yu et al.,BioTechniques 34:1008-1022, 2003) or, for example, a brush-like hydrogelsuch as the hydrogel disclosed in U.S. Pat. No. 6,994,964.

As noted above, the application of an electric potential differencebetween two electrodes separated by an electrolytic solution can resultin electrochemical reactions at the electrode-electrolyte interface ifthe applied voltage exceeds a threshold value. In such a case, gasbubbles may be generated at the electrodes due to electrolysis of wateror electrochemical reactions of electrolytic ions. The gas formation canrapidly obstruct the channel leading to disruption in normal flowcharacteristics. In addition, the pressure increase in the channel couldcause mechanical damage of the device. Finally, the products of theredox reactions due to Faradic currents may degrade the biologicalmolecules in the solution rendering the sample preparation unsuitablefor the downstream assays.

One method for avoiding such problems is the application of analternating voltage with sufficiently high frequency such that gasbubble formation is minimized as discussed above. In another approachthe generation of oxygen and hydrogen bubbles can be suppressed byadding a redox-couple to the sample flowing along the electrodes. As anexample, quinhydrone, which is a complex between hydroquinone (H₂Q)acting as an electron donor and p-benzoquinone (Q) acting as an electronacceptor, can be added to the flow streams. Instead of water oxidationand reduction that generates oxygen and hydrogen, now H₂Q is oxidizedand Q is reduced without any bubble generation. A drawback of bothforetold methods is that the interfacial electrochemical reactionsinvolving macromolecules cannot be fully avoided. It is further notedthat most macromolecules are charged in an aqueous medium, and thus maydrift to the interfaces under the influence of high fields typicallyused for the electrical lysis.

As noted above, the generation of gas bubbles and interfacialelectrochemical reactions can be avoided by providing a channel thatincorporates blocking electrodes to suppress a Faradic current. It iswell known, however, that the application of a constant electric fieldin a channel containing an aqueous ionic solution, where the channel isconfigured to suppress a Faradic current, results in formation ofelectric double layers near the electrodes which rapidly screen theapplied electric field in the inner regions of the channel. Accordingly,the actual electric field experienced by the suspended cells, referredto as the “effective field”, may drop to a small fraction of thenominally applied field shortly after the field is switched on.

In one embodiment, the aforementioned drawbacks associated withscreening of the electric field within the liquid are avoided asfollows. As noted above, the applied voltage is provided as a train ofamplitude modulated pulses. If the duration of the pulses does notexceed the characteristic charging time of the electrodes, then theshielding effects on the effective electric field can be tolerated.Accordingly, in one embodiment the electrodes (for example, electrodes 7and 8 in FIGS. 1-3) are provided as a thin conductive substrate and witha thin dielectric coating in contact with the channel fluid, where thesurface profile of the conductor and dielectric is microstructured forsurface area enhancement such that the blocking electrode surface areasubstantially exceeds the surface area of the corresponding flatsurface. The large capacitance thereby achieved enables a charging timegreater than one microsecond, such as on the order of tens ofmicroseconds.

The capacitance of the blocking layer can also be enhanced by providinga thin dielectric layer having a high dielectric constant. In oneexample implementation, the metal substrate is aluminum, and thedielectric layer is aluminum oxide (Al₂O₃). This aluminum oxide (Al₂O₃)dielectric layer is formed by electrochemically oxidizing the aluminum(anodized aluminum). In order to increase the effective surface by asmuch as 100 times and to provide a corresponding increase to thecapacitance per unit nominal area, the electrode is etched with a densenetwork of microscopic cavities and tunnels.

In the context of the present disclosure, these types of electrodes areidentified by the term SEOA (surface enhanced oxidized aluminum). Thethickness of the dielectric layer is determined by the applied voltageduring the electrochemical forming (anodizing) process. In one exampleimplementation, the thickness is chosen to be 2 nm per each volt thatcan be safely applied on the electrode. In examples for which theapplied voltages are about 200 V, the thickness of the dielectric layerfor safe operation under long duration pulses is thereby estimated to beon the order of 400 nm. This thickness can in some cases result in acharging time that is too short, thus reducing the duration of theeffective field in the channel to undesirably short periods. Inaddition, an appreciable amount of the applied voltage is dropped in thedielectric layer, which generally has much lower dielectric constant ascompared to water. In practice, when the duration of the pulses in thetrain, t_(p), do not exceed 1 ms, much lower dielectric thickness ofabout 50 nm can be used without the hazards of establishing Faradiccurrent due to onset of anodization processes on the electrode.Investigations by the inventors have indicated that an aluminum oxidelayer thickness of 50-200 nm is suitable for most applications describedherein. For example, in the case of the electrical lysis of Grampositive bacteria, electric fields of over 10 kV/cm are desirable, and athickness greater than 50 nm is desired to avoid electrical breakdown.In some embodiments, the dielectric thickness and surface areaenhancement may be selected to provide a capacitance in the range ofapproximately 0.5 μF/cm² to 200 μF/cm². In other embodiments, thecapacitance of the dielectric layer may be between approximately 2μF/cm² to 50 μF/cm². The selected dielectric constant may depend on theionic strength of the suspension liquid and the constraints of thefrequency response of the driving electronics. For example, when theionic strength of the liquid is below 1 mM and cost considerationsrequires the maximum operating frequency of the driving electronic toless than 10 kHz, a the capacitance may be chosen to be greater than 5pF/cm².

Although aspects of the disclosure are described with reference tosurface enhanced oxidized aluminum (SEOA), it is to be understood thatSEOA is merely one example material for implementing the presentembodiments. In another example, a substrate with a different metal(with high surface area) and an oxide layer may be employed, such astantalum and tantalum oxide. In another embodiment, silicon and silicondioxide may be employed, where the silicon may be doped with appropriateconcentration to provide suitable conductivity.

In one example embodiment involving a blocking electrode formed fromsilicon and silicon dioxide, the silicon may be porous silicon, such asmacroporous silicon or microporous silicon. In one example, the siliconis macroporous silicon with pore walls that are sufficiently thick tosupport the growth of an oxide layer with a thickness on the nanometerscale, while maintaining an underlying layer of conductive silicon.Suitable oxide layer thickness and surface enhancement are selected asdescribed above in the SEOA example.

In the preparation of porous silicon, the silicon substrate is typicallydoped, thereby providing a conductive electrode for use with the presentmethods. In some implementations, the porosity of the silicon can becontrolled by varying the etching conditions, and/or post-etching thestructure in a suitable etchant (such as hydrofluoric acid). The oxidelayer may be added after etching, where the oxide is generated byoxidation in a suitable thermal environment. As will be apparent tothose skilled in the art, the thickness of the oxide layer can becontrolled according to the time and temperature of the thermalincubation. The pores may be formed as an ordered array oftwo-dimensional pores, for example, by photolithographically definingpore nucleation sites, or the pores may be form as a disorderedstructure.

The electrical properties of the channel can be modeled by theequivalent electrical circuit presented in FIG. 5(A). The capacitanceC_(DL) corresponds to the dynamic double-layer capacitance at theinterfaces of dielectric layer and the liquid in the channel. R_(DL) isthe parallel (in the direction of the channel thickness) resistancecorresponding to leakage current in the double layer.

In general, values of C_(DL) for flat metal surfaces fall in the range5-50 μF/cm² depending on the type of electrode, ionic strength andcomposition of the solution, temperature and voltage. However, roughnessof the surface can increase the capacitance to higher values.Capacitance C_(DE) is the capacitance of the dielectric layer whosevalue depends on the layer thickness and the effective area of theelectrode. The capacitance for the SEOA electrodes used in theexperiments described herein was either 6 or 36 μF/cm². These electrodeswere designated as SEOA1 and SEOA2, respectively.

Resistance R_(DE) is the equivalent parallel resistance of thedielectric layer and accounts for leakage current in the capacitor. Itdecreases with increasing capacitance, temperature and voltage. Typicalvalues for R_(DE) are on the order of 100/C_(DE) MΩ with C_(DE) in μF.R_(CH) represents the bulk solution resistance and C_(CH) the bulkcapacitance. The value of C_(CH) is so small that it can be approximatedwith open circuit. For a channel with a height of 100 μm, the resistanceR_(CH) is about 200 S2 per cm² of the electrode, when the ionic strengthof the liquid is 0.5 mM. R_(LOAD) is the sum of the power supply outputresistance and the input resistance of the electrodes. All theelectrical parameter values, with the exception of R_(LOAD), R_(DE) andC_(DE) are dependent on the ionic strength of the carrier solution. Theload resistance modifies the voltage division among the circuitcomponents and becomes particularly important at higher ionic strengths.

Considering the typical values of the electrical parameters, theequivalent circuit can be simplified as presented in FIG. 5(B). Theresistances R_(DE), and R_(DL) are sufficiently large that they can beapproximated as open. The double layer capacitances and dielectric layercapacitances have been combined in series as C_(Eff). The double layercharging time, according to this circuit model, is given by

t _(c)≈(R _(LOAD) +R _(CH))(C _(DE) C _(DL))/2(C _(DE) +C _(DL))≡(R_(LOAD) +R _(CH))C _(Eff)   (1)

It is noted that this charging time is 1-2 orders of magnitude largerthan that achievable without surface enhancement of the electrode.Accordingly, the use of SEOA as one or more of the electrodes in thedevice provides a substantial increase in the charging time. Forexample, in one embodiment, the charging time may be at least onemicrosecond for liquids having an ionic strength below about 10 mM,thereby supporting the aforementioned methods of electrical lysis andsample treatment. FIG. 5(C) presents a typical current response of thechannel to an applied bipolar square pulse for electrodes SEOA1 andSEOA2. As is observed, the decrease in the current, which is anindicator of the effective field, is sufficiently small over the 50 μsduration of the square pulses. The channels, which were filled with 0.4mM phosphate buffer, had dimensions 28×3.17×0.1 mm^(3.)

An example method of employing the present device for sample processingis now illustrated with reference to FIG. 2. The external voltagesource, 20, applies a potential difference between the two electrodes, 7and 8, in the form of an amplitude modulated train of pulses. In oneexample, the pulses are bipolar square pulses. Other pulse shapes, whichmay ease the design of driving electronics, can be utilized. However,the published data on inactivating microorganisms in food samplesindicate that square pulses are more effective in terms of irreversibleelectroporation.

The electrical characteristics of the channel depend on the ionicstrength of the aqueous solution. Accordingly, in example embodiments,the selection of an appropriate applied voltage, pulse duration andpulse count may be performed based on a prior knowledge of the ionicstrength of the sample solution, or alternatively based on in-situelectrical measurements for setting the parameters of the pulse train.In one example, this may be done according to the feedback based on theelectrical current monitored by the meter 21 via the control feedbackloop 22 and the controller unit 23.

In some embodiments, the maximum channel temperature may be maintainedat the desired temperature during the residence time, while avoiding anextensive liquid to vapour phase change in the sample. This may be doneby controlling the temperature within the channel using the measuredcurrent as a feedback parameter, and providing this feedback parameterto the voltage controller 23. The temperature and/or the residence timefor effecting a desired change in the sample may be controlled, inresponse to the measured current, according to many possible methods. Inseveral non-limiting examples, the current (and/or a parameter relatedto the current, such as the impedance) may be employed to control thechannel temperature by controlling the voltage pulse train amplitude,pulse-density (the number of pulses with fixed duration in given timeinterval), duty cycle, and/or pulsewidth.

In some embodiments, the temperature dependence of the electricalconductivity of the sample fluid may be used to monitor the temperatureof the liquid. The electrical conductivity of aqueous fluids typicallyincreases with temperature at a rate, depending on the composition ofthe fluid, of approximately 2% to 3% per degree Celcius (R. B.McCleskey, J. Chem. Eng. Data 2011,56, 317-327).

FIG. 5(D) illustrates an example implementation of the electricalprocessing channel 20 of a microfluidic device, which shown incross-sectional view A-A in FIG. 5 e. Channel 220 is connected by inletand outlet channels 221 and 222 to inlet and outlet ports respectivelythrough which fluids of interest enter and exit from the electricalchannel. The electrical channel has top and bottom surface electrodes225 and 226 which allow the application of an electric field across thechannel by the external voltage source 227, which is capable ofsupplying an amplitude modulated train of voltage pulses. The deviceincludes valves 223 and 224 for opening and closing inlet and outletports to enable superheating. Alternatively, flow restrictions in theinlet and outlet ports and/or channels may be used to enablesuperheating of the liquid. In both cases, temperature control isaccomplished through feedback from the detection of electrical currentflowing across the channel.

The resulting electrical current flowing across the channel is measuredby a current monitor 228 and supplied to the voltage controller 229. Theapplied voltage amplitude may also be supplied to controller 229, asrequired, for example, for measurement and subsequent control ofelectrical channel impedance. In one embodiment, the voltage pulses areregulated by controller 229 in response to current and voltage feedbackto maintain electrical channel impedance at a desired level. In anotherembodiment, the current monitor can be used to measure current duringinitial operation of the electrical channel and the controller 229 canbe used to determine the appropriate subsequent voltage train parametersin response to the measured current. The latter embodiment may be used,for example, to determine appropriate voltage train parameters forfluids of unknown ionic strength.

FIG. 5(F) illustrates an example embodiment of voltage controller 229.Voltage controller 229 may include one or more processors 330 (forexample, a CPU/microprocessor), bus 332, memory 335, which may includerandom access memory (RAM) and/or read only memory (ROM), one or moreinternal storage devices 340 (e.g. a hard disk drive, compact disk driveor internal flash memory), a power supply 345, one more communicationsinterfaces 350, external storage 355, a display 360 and variousinput/output devices and/or interfaces 355.

Although only one of each component is illustrated in FIG. 5(F), anynumber of each component can be included in voltage controller 229. Forexample, a computer typically contains a number of different datastorage media. Furthermore, although bus 332 is depicted as a singleconnection between all of the components, it will be appreciated thatthe bus 332 may represent one or more circuits, devices or communicationchannels which link two or more of the components. For example, inpersonal computers, bus 332 often includes or is a motherboard.

In one embodiment, voltage controller 229 may be, or include, a generalpurpose computer or any other hardware equivalents. Control andprocessing unit 325 may also be implemented as one or more physicaldevices that are coupled to processor 330 through one of morecommunications channels or interfaces. For example, control andprocessing unit 325 can be implemented using application specificintegrated circuits (ASICs). Alternatively, control and processing unit325 can be implemented as a combination of hardware and software, wherethe software is loaded into the processor from the memory or over anetwork connection.

Voltage controller 229 may be programmed with a set of instructionswhich when executed in the processor causes the system to perform one ormore methods described in the disclosure. Voltage controller 229 mayinclude many more or less components than those shown.

As described above, the voltage pulse train applied across the channelelectrodes will cause substantial current to flow in the channel fluidcausing rapid Joule heating of the fluid. The thermal behavior of theelectrical channel is influenced by a number of factors including theamplitude, frequency and duration of the applied voltage pulses, thethermal properties of the channel, the ionic concentration of thesolution, and the pressure in the channel.

Active feedback control of the voltage pulse train applied to thechannel is described by the example implementation of the controldiagram shown in FIG. 5(G). As the temperature of the fluid in thechannel is represented by the electrical impedance of the channel,impedance Z(t) obtained from measured voltage V(t) and current 1(t) isused as the feedback signal. An error parameter e(t) is obtained fromthe impedance relative to a predetermined setpoint impedance Z_(SP) andprovided to the controller.

It is to be understood that a wide range of choices exist forcontrolling the voltage pulse train in response to the error signale(t). In some non-limiting examples, the voltage pulse train amplitudemay be modulated, an inter-pulse delay may be introduced and modulated,or pulses may be skipped in relation to the magnitude of the errorsignal. In the latter two cases the voltage amplitude is held constantand hence the feedback signal may more simply be the measured current.For example, as shown in Example 6 below, the limiting case of pulseskipping was employed, whereby the voltage pulse train was halted when apredetermined current level was reached.

Active feedback control is now described in an example implementation inwhich voltage amplitude is modulated to reach and maintain predeterminedminimum impedance. An initial voltage pulse train of constant amplitudeand frequency is applied and upon reaching a predetermined setpointcurrent level, active feedback control is activated. Thereafter thevoltage controller adjusts the voltage pulse amplitude in response tomeasured electrical impedance in order to maintain the impedance at apredetermined setpoint level for the remainder of the voltage pulsetrain.

The performance or effectiveness of the electrical lysing and electricaltreatment methods may be enhanced by increasing the maximum temperaturereached in the channel fluid and/or by increasing the residence time ofthe channel at elevated temperatures. The peak temperature which can bereached by the fluid in the channel is limited to the boilingtemperature of the fluid, which is influenced by bubble nucleationconditions and pressure. Since the fluid temperature in the channel isgenerally non uniform and greater in a central region, bubble nucleationmay be delayed until fluid temperatures in this region have exceedednormal boiling temperature. Further increases in the boiling temperaturecan be achieved when the pressure in the channel is greater thanatmospheric pressure.

When fluid temperatures are achieved which are greater than the boilingtemperature at atmospheric pressure, the fluid is said to besuperheated. This can be done actively by pressurizing the channel priorto or during the application of voltage pulses. Alternatively, byclosing the inlet and exit ports of the channel, increased pressure canbe developed in the channel as the temperature of the fluid is increasedduring the application of voltage pulses. Increased pressure in thechannel can also be achieved with open inlet and outlet ports byproviding features at the inlet and outlet to the channel which restrictfluid flow.

In the latter case, as the voltage pulses are applied, the temperatureof the fluid increases, causing expansion of the fluid and ultimatelythe generation of a gas phase when the fluid reaches its boilingtemperature. During the application of the voltage pulses, the channelentrance and exit flow restrictions cause a pressure drop between theelectrical channel and the exit to atmosphere which results in elevatedpressure in the electrical channel, leading to superheating of thefluid. Various mechanisms for providing flow restrictions are known bythose skilled in the art and include choosing appropriate electricalchannel inlet and outlet orifice size and geometry and/or providinginlet and outlet channels for which the hydraulic diameter, length,surface roughness and the addition of bends are chosen so as to increasepressure drop along its length during fluid flow. Residence times canalso be effectively increased using this method by slowing the formationof the gas phase, which results in the lower rate of decrease of currentfollowing the peak in the current response of the channel, as can beseen in the example presented below (FIG. 19A).

The current or impedance feedback control methods described previouslyprovide a means to control the maximum temperature in the channel and/orthe residence time at predetermined temperatures. When used inconjunction with an electrical channel with closed or restricted inletand outlets, as described above, superheating of the fluid can beachieved and monitored via the electrical current and/or impedance ofthe channel.

It may be desirable under these superheating conditions to to limit thepressure buildup in the channel. This may be achieved, for example, byhalting the delivery of voltage pulses or by modulating the electricalenergy delivered to the electrical channel once a predetermined setpointimpedance is reached.

Channels with minimal or no flow restriction at the inlet and outlet,herein termed open channels, may also be so controlled, albeit at lowertemperatures, prior to the onset of liquid to vapour transition. Longerresidence time at elevated temperatures may be achieved by activecontrol of the voltage pulse train in response to measured impedance insuch way as to maintain predetermined minimum impedance.

In addition to increasing the exposure time of cells and macromoleculesto elevated temperatures, longer residence times also allow time forheat transfer to occur from hot central zones in the fluid to the coolerboundary regions thus exposing more cells and macromolecules to thehigher temperature levels.

Upon applying the voltage to the electrodes, the passage of the ioniccurrent through the sample liquid results in the generation of heatduring the pulse train which is followed by rapid cooling after thepulse train is ceased. As it will be seen in the experimental examplespresented below, the performance of the channel during electricaltreatment depends on its thermal characteristics, and the channelgeometry, dimensions, and thermal material properties are selected to besufficiently thermally insulating to support flash heating under theapplication of voltage pulses to the electrodes (for example, such thatthe temperature of the liquid increases at a rate in excess ofapproximately 250 degrees Celsius per second), while also beingsufficiently thermally conductive to provide for rapid, sub-secondcooling time after removing the applied voltage pulses. In other words,by selecting channel materials with an appropriate thermal conductivity,and channel dimensions and geometries that provide an appropriate heatcapacity (or, equivalently, an appropriate thermal mass or heat sinkingcapability) for the surface to volume ratio of the channel, the initialheat rise can be followed by a rapid cooling cycle. It will beunderstood that there are many different configurations of the channeldimensions and geometry and choices for the channel materials that willexhibit a suitable thermal response. Accordingly, the specific examplesprovided herein, and in the examples below, are provided as heuristicand non-limiting examples. Other configurations and material choices maybe made by routine experimentation without departing from the scope ofthe present disclosure.

The thermal properties of the channel are dependent on many differentchannel parameters. For example, the channel conductivity and heatcapacity can be controlled according to the geometry and/or thickness ofthe metal electrode. Most electrodes will have a high thermalconductivity, but the thermal properties of the channel can be tailoredby selecting an appropriate electrode thickness to provide a suitableheat capacity and an appropriate (e.g. thermally insulating) substrateupon which the electrodes are supported.

Accordingly, one or more of the channel electrodes may be provided as ametal foil or coating having a high thermal conductivity and/or a highheat capacity relative to the total volume of fluid in the channel topromote rapid cooling after electrical treatment, while at the same timeproviding a sufficiently small heat capacity such that the flash heatingcan produce a rapid temperature rise within the channel during theapplication of the voltage pulses (for example, a temperature risegreater than approximately 250 degrees Celsius per second).

Alternatively or additionally, the channel may include lateral heatsinking elements. In one embodiment, the thermal conductivity of theside walls of the channel may be selected such that the side walls canact as a lateral heat sink. In another example, one or both electrodesmay extend beyond the channel-defining region, thus providing anotherlateral heat sinking path.

The electrical and thermal behavior of the channel is now illustratedwith an example of measurements taken during a typical channelexperiment. FIG. 6 shows the envelope of the measured electrical currentpulses in a channel whose inlet and outlet ports were open toatmospheric pressure and which was subjected to an applied voltage inthe form of a square bipolar pulse train with n=800 pulses, an amplitudeof 196 V and a frequency of 10 kHz (the load resistance, R_(LOAD) inFIG. 5B, was 10 Ohm). The channel structure comprised an aluminum upperplate, glass lower plate, with an inner surface of each plate includinga 0.1 mm polyimide insulating layer, and with a SEOA electrode (SEOA1)provided on each polyimide insulating layer, such that the oxidizedelectrodes contact a fluid within the channel, and with a 0.1 mmpolyimide spacer defining the channel height.

The channel with dimensions 28×3.17×0.1 mm³ was filled with 0.4 mMphosphate buffer. The characteristic feature of the channel electricalcurrent response is the initial monotonic rise of current to a maximumvalue, followed by a rapid reduction to a minimum value at time t_(c),accompanied in some cases by quasi periodic fluctuations as seen in FIG.6.

Because, in general, the conductivity of an aqueous solution is alinearly increasing function of temperature (Aqueous Systems at ElevatedTemperatures and Pressures, Chapter 10, Elsevier 2004), one may concludethat the initial rising current is due to Joule heating of the fluid inthe channel. Since the channel is open to the atmosphere, one wouldexpect that the fluid temperature will be limited to the liquidsaturation state temperature at atmospheric pressure which for thesolution used is approximately 100° C. Without intending to be limitedby theory, it is believed that further energy input will result in aphase transition occurring in those areas of the channel which havereached this temperature. The presence of vapor in the channel, as aresult of the phase transition, will significantly reduce theconductance of the channel, leading to the observed decrease of current.

A finite differences analysis of transient heat transfer in the channelwas used to estimate channel temperatures both during and following theelectrical pulse train. Fourier's law of heat conduction was solvednumerically in conjunction with conservation of energy for a channelspatially discretized across the thickness and the width of the layeredchannel assembly. Joule heating of the channel fluid is calculatedassuming a linear thermal dependence of the conductance of the fluid thelinear parameters of which are determined from the experimentallymeasured current at the initiation of electrical pulses and theiterative solution of the current at 100° C. which lies on theexperimentally measured curve of FIG. 6. Thereby the onset of phasechange is estimated to occur at the center of the channel at t=0.024 s,as identified in FIG. 6.

The simulated spatial temperature distribution in the channel at t=0.024s is provided in FIG. 7. FIG. 8 shows the calculated time dependence ofthe spatial maximum temperature, assuming that the applied voltage isset to zero at t=0.024 ms. In this model, if the voltage remains appliedfor a further time t_(r), the peak temperature will be maintained andthat portion of the fluid which reaches 100° C. will undergo a phasetransition.

The existence of a mixed liquid-vapour phase that occurs following theinitiation of the phase transition is believed to result in a localizedreduction of conductivity, which in turn leads to a redistribution ofcurrent within the channel. It is further believed that after the onsetof the phase transition, the mixed phase region undergoes expansion dueto the continued increase in fluid temperatures outside the region ofvaporization. Accordingly, as the mixed phase region expands further,the current within the channel is expected to decrease substantially asnet conductivity of the channel falls.

Referring now to FIG. 6, the time dependent current profile appears tobehave in a manner that is consistent with the interpretation providedabove. Specifically, the current initially rises towards a maximum valueas the phase transition is expected to be initiated, after which thecurrent decreases as predicted by the mixed-phase model.

Furthermore, the quasi-periodic features following the maximum currentmay be attributed to further phase transition cycles, with correspondingincreases and decreases in conductivity and current. These phasetransition cycles are believed to be a result of the reduction incurrent and subsequent cooling and condensation, which in turn leadsagain to a rise of current and another cycle of vaporization andcondensation.

The temperature of the channel fluid is expected to be in a quasi steadystate during the phase transition cycles. As a result, this mechanismmay provide passive control of the peak temperature during electricalprocessing. This constitutes a self-limiting passive feedback mechanismfor the temperature control.

In another example implementation, the current flowing between thedevice electrodes may be monitored by identifying an initial peak in thetime-dependent current (which may correspond to the onset of a phasetransition in the liquid), and continuing to apply to voltage pulses fora prescribed time duration after the detection of a reduction in currentbelow the peak. The current may also or alternatively be monitored todetect the presence of one or more features in the time-dependentcurrent, such as for example local current minima or maxima, and theapplication of the voltage pulses may be maintained during a timeinterval corresponding to one or more of these features.

Referring again to FIG. 8, the simulations indicate that following theremoval of the voltage, the channel fluid cools down rapidly due tothermal diffusion from the relatively small volume of channel fluid tothe neighboring materials of the flow cell, returning close to initialtemperatures in less than 1 second. This is a useful mechanism that canbe taken advantage of to avoid thermal damage to macromolecules arisingfrom prolonged exposure to high temperatures.

In another embodiment, the pressure within the channel may be controlledin order to superheat the fluid within the channel during electricaltreatment. For example, the channel may include valves for enclosing theinternal volume, thereby increasing the pressure within the channel inresponse to an electrically induced increase in temperature.

FIG. 9 shows the envelope of experimentally measured electrical currentpulses in a channel having a configuration as described above, butfurther including a shutoff valve at the inlet and the outlet port. Thechannel was subjected to an applied voltage in the form of a bipolarsquare pulse train with n=400 pulses, an amplitude of 200 V and afrequency of 10 kHz. As can be seen in FIG. 9, when the channel inletand outlet ports are closed, the current initially follows the behaviorof the open port channel, but instead of reaching a maximum value with asubsequent decrease of current, the current monotonically increasesuntil the end of the pulse train.

Accordingly, during electrical heating, pressure in excess ofatmospheric pressure builds up in the closed channel, such that thechannel fluid remains in a liquid phase and becomes superheated as itstemperature exceeds the atmospheric liquid saturation state temperature.Estimates obtained from the finite differences thermal analysis suggestthat the confined liquid in the closed channel reaches temperatureswhich are on average approximately 35° C. higher than the open channelin this example. The thermal analysis also indicates that after removingthe applied voltage, the superheated fluid rapidly cools, returningclose to initial temperatures within 1 second.

By regulating the pressure in the channel, the superheated fluidtemperature can be controlled to limit the peak temperature and/or tomaintain a temperature near the peak temperature. More specifically,when the pressure is maintained at a fixed value, the continuedapplication of voltage pulses after reaching the peak current value(i.e. near the time of phase transition initiation) will maintain aquasi-steady state in which the current, temperature and conductivitymay oscillate within a range of values.

Referring to FIG. 10, an example embodiment of a channel device isillustrated which incorporates such a pressure regulation mechanism (forexample, an active pressure regulation mechanism, or a passive pressureregulation mechanism). The device, similar in other respects to devicesdescribed earlier, has a shutoff valve 31 at the inlet port 3 and ashutoff valve 32 at the outlet port 4 section of the channel. Inaddition, a pressure regulation mechanism 30 is in fluid communicationwith the channel 2 between the inlet and outlet shutoff valves. Thispressure regulation mechanism can be of the form of any such backpressure regulators or pressure relief valves well known in the art andis represented here by a spring loaded plunger and expansion cavity.

In one example embodiment, the pressure regulation device is aspring-loaded plunger or membrane where the spring is preloaded suchthat the regulator activates at a pre-determined minimum pressure whichcorresponds to the desired superheated fluid temperature. When thisminimum pressure is reached in the channel as a result of heating of thefluid, the plunger will move into the expansion chamber, expanding theeffective channel volume in response to a further increase in pressure.If the spring constant is sufficiently low and a sufficiently largeexpansion cavity is provided, the pressure will be maintainedapproximately at a constant peak value and a saturated liquid state willbe achieved in the fluid when a requisite amount of energy is suppliedto the channel. With further energy input a phase transition will occurin the region of the channel which has achieved the liquid saturationstate and a mixed liquid-vapour phase will be generated at approximatelya constant temperature.

In an alternate embodiment the spring may have no pre-force and thespring stiffness may be chosen such that the pressure increases withtemperature but at a rate which allows the liquid to reach saturation ata pre-determined temperature. Thus the resulting temperature will not beconstant but will be controlled or will be responsive to applied voltagein a pre-determined manner.

An example of this later embodiment is a membrane of pre-determinedstiffness either as part of the whole of one or both of the channelwalls or one or more of the walls of a sealed cavity in fluidcommunication with the channel. A further example of this embodiment isa cavity or channel which is separated from the main channel by agas-permeable membrane allowing the transport of gas but not liquid.Thus the compressibility of the air or gas will provide the complianceto control the pressure in the manner described.

In yet another embodiment, the device may include an active pressureregulation mechanism that is externally controllable. Such an activepressure regulation device may be controlled such that the pressure isregulated within the channel in synchronization with the timing of theapplied voltage. In another example, the device may include both acontrollable pressure regulation device and a pressure sensor, where thepressure sensor signal is provided as an input (feedback) signal to acontroller, and where the controller is interfaced with the pressureregulation device for controlling the pressure within the channel.

In general, the device can be designed to operate over a wide range ofionic strengths. However, high ionic strength, such as ionic strengthabove approximately 1 mM, generally requires circuitry capable ofdelivering higher currents. This results in higher electrical powerrequirements with its added complications. Therefore, reducing the ionicstrength may be performed prior to the application of the voltage.

The reduction in ionic strength may be readily accomplished in caseswhere the sample liquid includes cells by filtering the cells as shownin FIG. 3 and FIG. 4 and flowing a low-ionic strength liquid into thechannel prior to electrical processing. FIG. 4(C) provides a flow chartthat illustrates a method of performing electrical processing of cellsafter initially capturing the cells on a filter as described in FIG. 4.In step 100, a sample liquid is flowed (such as from a first port 3 to asecond port 34 in FIG. 4), through the filter (such as filter 33 in FIG.4). Cells within the liquid sample are captured by the filter, asdescribed in step 105. After having captured the cells an additionalliquid is optionally flowed through the filter to wash the cells andfluid paths of the sample fluid, as in step 110. Then a fluid suitablefor performing electrical lysis and treatment and optionally appropriatefor downstream processes is flowed to resuspend the cells and carry themto the electrical channel as in step 115. After having carried the cellsto the electrical channel suspended in this liquid, the electricalprocessing may be performed, as shown in step 120. Then the lysate maybe extracted by flowing the additional liquid through the channel andcollecting the outflow as in step 125. After performing the electricalprocessing step, the ionic strength of the additional liquid residingwithin the channel may be increased. For example, this may be achievedby adding a salt-containing reagent to the additional liquid, or, inanother example, by flowing the additional liquid through an additionalchannel containing dried salts that can be dissolved to achieve thedesired ionic strength.

In another embodiment, ion reduction in the sample can be performed bythe sample cleanup module 65 of FIG. 11 that may be integrated in theinlet port 3 of the device (see FIG. 1) or in a chamber fluidicallyconnected upstream of the inlet port. The sample cleanup module, 6,consists of inlet 60, outlet 61, an optional pre-filter, 62, packed ionexchange resins, 63, and a filter, 64. The optional pre-filter 62excludes large particles such as cationic exchange resins and non-ionicadsorbing resins, which are employed in some samples such as the bloodculture media of the Becton Dickinson blood culturing system.

The ion exchange resins (63) include mixed cationic and anionic resinswhich serve to deionize the sample, and to optionally capture smallerionic particles that may be present in the sample. In the case of bloodculture samples, some blood culture media includes activated charcoaland fuller's earth powder, for example, as in the BioMerieux system. Thefilter 64 retains the ionic resins and bound ions and ionic particles toprevent them from entering the device.

For the deionization of ions and ionic particles, mixed H⁺ form cationexchange resin and OH⁻ form anion resin may be used. In a specificexample involving Na⁺ and CL⁻ ions in solution, Na⁺ in the medium bindsto the cation resin in exchange of H⁺ and Cl⁻ binds to the anion resinin exchange of OH⁻. Removed H⁺ and OH⁻ will form water molecules. Thismethod is widely applied in water deionization applications. In oneexample implementation, microporous gel resins with the pore size largerthan the size of bacteria may be used. In addition, in applicationsinvolving bacteria, negatively charged bacteria can still bind to thesurface of the anionic resin and nonspecifically bind to the surface ofthe resins, and this may be prevented by treating both types of resinswith a non-ionic surfactant such as TritonX-100. Examples of mixedresins are Amberlite MB-150 from Rohm & Hass and Dowex-Marathon MR-3from Dow Chemicals with particle sizes ranging from 500-700 μm.

The operation of the ion reduction device is henceforth described byreferring to FIG. 3, as applied for treating a urine sample containingbacterial cells for obtaining an assay-ready lysate for a 16S rRNAhybridization assay. Once the entire sample liquid has passed throughthe channel and the suspended cells are retained on the membrane filter(i.e. concentrated on the filter) an optional washing step can beperformed by injecting a washing liquid into the channel. The flow ofthe washing liquid carries away excess ions (e.g. this step achievessample cleanup). The channel may then be filled with a low ionicstrength liquid, such as a 0.5 mM phosphate buffer.

The release of cellular contents is accomplished by applying a pulsetrain to the electrodes, thereby causing the electrical lysis. Amongstthe released cell contents are ribosomes that contain 16S rRNA entangledwith ribosomal proteins. In one embodiment, the electrical pulses areapplied such that the combined action of the persisting pulses and flashheated medium untangles/denatures rRNA from the proteins, possibly bydenaturing the ribosomal proteins. Furthermore, flash heating of mediummay also play a role in achieving a re-conformation of the rRNA moleculethat is appropriate for hybridization assays. Moreover, the RNAseenzymes may also be deactivated via the electrical treatment mechanismdescribed above involving electric field effects and flash heating.Finally, a pressure differential may be applied to the channel todeliver the lysate through the outlet port to a downstream chamber wherean assay may be performed. Prior to performing a hybridization assay,the ionic strength of the lysate may be increased to a suitable level.As noted above, this may be achieved by adding a salt-containing reagentto the lysate, or, in another example, by flowing the lysate through achannel containing dried salts that can be dissolved by the lysate toachieve the desired ionic strength.

As noted above, the present devices and methods may be employed for awide range of diagnostic methods and other sample processingapplications. In some example applications, the electrical lysis andelectrical treatment steps may be performed in a single step, where acell is lysed and the lysate is treated. Alternatively, electrical lysismay be initially performed, where the electrical parameters of theelectrical lysis steps are selected to provide efficient lysis.Electrical treatment of the lysate may then be provided in a subsequentstep, where the electrical parameters of the electrical treatment stepare chosen to provide efficient or sufficient electrical treatment.

In other example implementations, the sample may be delivered to thechannel in the form of a lysate based on a preceding or separate lysisstep. Alternatively, the sample may contain molecules that are to betreated according to the aforementioned electrical treatment methods,but where the molecules may not originate from a preceding lysis step.

In one example implementation, the devices and methods may be employedfor applications involving the rapid lysis of bacteria and thepreparation of the lysed bacteria for PCR. In particular, the presentmethods may be employed as a sample preparation method for colony PCR,in which PCR is employed to screen transformed bacterial colonies withsuccessful incorporation of a gene insert into a plasmid. The presentelectrical treatment methods may be employed to denature or deactivatePCR inhibitors and/or contaminants, such that PCR may be performedwithout performing a previous nucleic acid extraction or purificationstep. For example, bacteria obtained from a bacterial colony may besuspended in a low ionic strength buffer and provided to a device asdescribed above, such that the buffer is flowed into the channel. Asuitable voltage pulse train is then applied to obtain lysis andelectrical treatment of the lysate (example suitable values are providedin the preceding disclosure). The processed lysate may then be directlymixed with the appropriate PCR reagents for performing direct PCR.

In another example application, the present electrical treatment methodmay be employed for the denaturing of enzymes used for nucleic aciddigestion or modifications. The present devices and methods may beemployed for the electrical treatment of a sample in order to denatureor deactivate the enzymes. It is to be understood, however, that theeffectiveness of this method may be limited by the ionic strength of thesample, and the ability to employ ion exchange resins for thedeionization of samples. The electrical parameters required for aspecific enzyme inactivation without compromising the integrity of thenucleic acid may be determined by performing a series of experiments.

Suitable values for the various parameters employed in electrical lysisand treatment will depend on the properties of the liquid, cells, andmacromolecules of interest that are to be processed in the device.Suitable values may also depend on the application. For example, in someapplications, in may be preferable to lyse a cell and releaseintracellular macromolecules (such as nucleic acids or proteins) withoutcausing substantial denaturing or degradation. In such a case, anelectrical processing protocol may be preferred in which the thermalenvironment, electric field, and timescale of treatment are chosen tolyse the cells without denaturing or degrading the macromolecules.

In other applications, may be preferable to lyse a cell and releaseselected intracellular macromolecules (such as nucleic acids) whilecausing substantial denaturing or degradation of other intracellularmacromolecules that are released (such as a nuclease). In such a case, adifferent electrical processing protocol may be preferred in which thethermal environment, electric field and timescale of treatment arechosen to lyse the cells and to denature or degrade the othermacromolecules.

Generally speaking, the following ranges may be employed for electricalprocessing of cells. The electric field strength within the channel thatis produced by the application of the voltage pulses may range betweenapproximately 200 V/cm<E<50 kV/cm, depending on the type of cell that isto be processed and the degree of electrical processing that is desired.A range of approximately 2 kV/cm<E<30 kV/cm may be preferable for lysisof microorganisms and using the lysate for performing diagnostic testson the released nucleic acids.

According to different example implementations, the pulse width ofindividual voltage pulses may range between approximately 1 μs<t_(p)<10ms, depending on the type of cell that is to be processed and the degreeof electrical processing that is desired. A range of approximatelyt_(p)<1 ms may be preferable for avoiding the electrical breakdown ofthe dielectric coating in the case of blocking electrodes, minimizingthe accumulation of the electrochemical products in the case ofnon-blocking electrodes. A range of approximately t_(p)>10 μs ispreferred for lowering the high frequency demands of drivingelectronics.

According to other example implementations, the time duration over whichthe voltage pulses are applied may be less than about 5 s, depending onthe type of cell that is to be processed and the degree of electricalprocessing that is desired. In some cases, such as to minimize the heatinduced degradation of target macromolecules and decrease the powerdemands of the driving electronics, an effective time duration forelectrical processing may be less than about 100 ms.

According to other example implementations, the ionic strength of thecell containing liquid may range from approximately 0.1 mM<I<100 mM,depending on the ionic composition of the initial sample that is to beprocessed and the degree of electrical processing that is desired. Insome cases, when filtering is used and fluid exchange is allowed, a moresuitable range for the ionic strength may be from approximately 0.1mM<I<10 mM, or 0.2 mM<I<1 mM.

According to other example implementations, the peak temperature of theliquid within the channel during the application of voltage pulses mayrange from approximately 30° C.<T_(p)<250° C., depending on the type ofcell that is to be processed and the degree of electrical processingthat is desired. In some applications, such as lysis of microorganismsand using the lysate for performing diagnostic tests on the releasednucleic acids, it may be preferable for the temperature range to liewithin approximately 80° C.<T_(p)<200° C.

The heating rate of the liquid for the electrical processing may begreater than approximately 250° C./s, depending on the type of cell thatis to be processed and the degree of electrical processing that isdesired. In some cases, such as lysis of Gram positive bacteria, fungi,and spores, a suitable range may include rates greater than about 2000°C./s.

The cooldown time of the liquid following electrical treatment may beless than approximately 1 s, depending on the thermal sensitivity of thetarget macromolecule. In some cases, such as when the targetmacromolecule is particularly sensitive, a preferred range may includetimes below about 100 ms.

As described above and in the forthcoming examples, the present devicesand electrical processing methods may be employed for the preparation ofa wide variety of sample types. In many cases, a wide variety of celltypes may be processed with the same device properties, but withdifferent electrical parameters and/or pressure regulation duringelectrical processing. Specific types of cells are considered brieflybelow.

The preceding ranges of parameters are provided as examples and are notintended to limit the scope of the disclosure. It will be understoodthat those skilled in the art, aided by the present disclosure, mayidentify additional suitable ranges or combinations of parameters byroutine experimentation.

In the examples which follow, the pulse train consisted of a number, n,of bipolar square pulse cycles with a frequency of 10 or 20 kHz. Threetypes of channels with the following dimensions (H×W×L) were utilized:0.1×6.4×28 mm³ (wide-long, with a volume of about 18 μl), 0.1×6.4×16 mm³(wide, with a volume of about 10 μl) and 0.1×3.2×28 mm³ (narrow, with avolume of about 9 μl). The sets of pulse amplitude, V, the trainduration, t, and the ionic strength are known as test parameters in thecontext of this disclosure.

The following examples are presented to enable those skilled in the artto understand and to practice embodiments of the present disclosure.They should not be considered as a limitation on the scope of thepresent embodiments, but merely as being illustrative and representativethereof. The examples may also serve to provide example and/or suitableranges of parameters that for the operation of the device in variousapplications.

EXAMPLE 1 Deactivation of Enzymes

The experiments described in this example are intended to demonstratethe effects of electrical and thermal parameters on the capacity of thedevice for altering the structure and function of proteins bydemonstrating deactivation (complete or partial) of selected exampleenzymes. The experiments demonstrate an aspect of the electricaltreatment function and capability of the devices disclosed herein,involving the modification of protein conformation.

A mixture of three enzymes, glucose-6-phosphate dehydrogenase (G6PDH)(G-8629, Sigma) 1 unit/mL, beta-glucuronidase (G-7396, Sigma) 100units/mL and horseradish peroxidase (HRP) 1:500,000 dilutions (A-0168,Sigma) were prepared in 0.1 and 0.4 mM phosphate buffer pH 7.4. Thesample volume of 200 μL was passed through a narrow channel in steps of5 μL at intervals of 10 s during which a single pulse train, withamplitude of 150 V and frequency of 10 kHz, was applied.

The treated sample was tested for the enzyme activity compared to theuntreated sample. The enzyme activity was measured using the respectivesubstrates; glucose-6-phosphate (G6P) 6.6 mM, nicotinimide adeninedinucleotide (NAD) 4.0 mM, sodium chloride 90 mM, bovine serum albumin(BSA) 1%, sodium azide 0.09% in Tris 20 mM pH 5.0 for G6PDH,4-Nitrophenyl β-D-glucuronide (N-1627, Sigma) 1 mM in 50 mM phosphatebuffer pH 7.4 for Glucuronidase and 3, 3′,5 ,5′-Tetramethylbenzidine(TMB) followed by stop solution for HRP. The absorbance was measured at340 nm, 405 nm and 450 nm respectively.

The current envelopes corresponding to different ionic strengths arepresented in FIG. 12(A). The residual enzyme activities, for differentcombinations of pulse train durations and ionic strengths are presentedin FIG. 12(B). As it is observed the enzyme activity undergoes a drasticreduction for train durations exceeding the critical duration, t_(c), ofabout 40 ms. The results suggest that the residence time after t_(c)does not have a pronounced effect on the enzyme activity reductionbeyond this time duration. In the case of lower ionic strength of 0.1mM, even though the critical time t_(c) at approximately 70 ms has beenpassed, the activity reduction is moderate, implying a possiblecorrelation between heating rate and enzyme deactivation.

EXAMPLE 2 Electrical lysis of Escherichia coli

The experiments described in this example are intended to demonstratethe effects of the electrical parameters on the efficiency of the devicefor lysing E. coli cells. NEB 5-alpha competent E. coli cells,transformed with pUC19 plasmid which contains ampicillin resistance geneand beta-galactosidase gene, were grown on LB agar plates supplementedwith 100 μg/mL Ampicillin, 60 μg/mL X-gal and 0.1 mMisopropylthio-β-D-galactosidase (IPTG). A single blue colony of E. coliwas cultured in LB broth supplemented with 100 μg/mL Ampicillinovernight at 37° C. The cells were centrifuged at 7000 rpm for 5 min.The cell pellet was washed twice and re-suspended in 0.1 to 0.4 mMphosphate buffer pH 7.4 at a concentration of 0.5 to 1×10⁹ CFU/mL.

To perform electrical lysis, 200 μL of the sample was passed through achannel in steps of 5 μL every 10 s. Then, three different analyticaltests were performed for assessing the efficiency of cell lysis; totalprotein assay, quantitative total nucleic acid assay, and 16S rRNAhybridization assay.

Total protein released in the cell lysate was assayed by BradfordReagent (B-6916, Sigma). The cell lysate was centrifuged at 7000 rpm for5 min and 50 μL of the supernatant was mixed with an equal volume ofBradford protein assay reagent. The color development was measured byspectrophotometer at absorbance 595 nm. As a reference cell lysiscontrol, cells were lysed mechanically by beating with an equal volumeof glass beads (106 μm, Sigma) for 2 min and the supernatant of glassbead cell lysate (GB) was assayed after centrifugation. The proteinconcentration was calculated by referring to the dose response curve ofFIG. 13, which has been prepared by running Bradford assay on differentconcentrations of BSA.

To measure the released nucleic acid quantitatively, the cell lysate wascentrifuged at 7000 rpm for 5 min and 50 μL of the supernatant was mixedwith an equal volume of SYTO-9 nucleic acid stain (S-34854, Invitrogen),2.5 μM concentration in 0.1 mM phosphate buffer pH 7.4. The fluorescencesignal was measured by fluorospectrophotometer at excitation wavelength485 nm and emission wavelength 515 nm.

Bacteria-specific 16S rRNA in the cell lysate was detected by asolid-phase sandwiched nucleic acid hybridization assay. Although totalcell lysate can be used as an assay-ready sample, to demonstrate theefficient release of rRNA out of the cells, the supernatant of the celllysate was used in the assay. The cell lysate was centrifuged at 7000rpm for 5 min to collect the supernatant containing released rRNA. Thesupernatant of 50 μI volume was added to Immobilizer Amino plate (Nunc)assay wells, in which 5 μM of biotinylated capture probe and 20 μg/mLstreptavidin (R1) were immobilized by spotting and non-specific bindingsites were blocked with 0.2% BSA and 0.1% Tween-20 in PBS pH 7.4. R2reagent, 0.2 μM FITC-conjugated detector probe in 1 M phosphate bufferpH 7.4, of 50 μL volume was added to the assay well and incubated at 55°C. for 20 min. After washing, 100 μL of R3 reagent, 1:1000 dilution ofHRP-conjugated anti-FITC antibody in the blocking buffer, was added tothe well and incubated at room temperature for 10 min. TMB substrate of100 μL volume was added after washing followed by addition of the stopsolution. The color development was measured by spectrophotometer at 450nm wavelength.

Example 2.1 Effects of Pulse Amplitude on Electrical Lysis Efficiency

The bacteria suspension of 5x10⁸ CFU/mL in 0.1 mM phosphate buffer pH7.4 was passed through a narrow channel in steps of 5 μL/10 s and 10 kHzpulse trains with different durations and amplitudes were applied to thesuspension. The duration, t, and amplitude, V, of any two differentparameter sets, (t₁, V₁) and (t₂, V₂), were related by t₁/t₂=(V₂/V₁)² toensure nearly equivalent electrical power delivery to the channel forboth cases. Moreover, besides selecting low ionic strength of 0.1 mM,the durations were chosen short enough not to heat up the liquid muchabove the biological temperatures. The average temperature the channelachieved, Tm, was estimated and recorded on the legend of FIG. 14(a),which illustrates the current envelope for different test parameters.

The results of 3 analytical assays; detections of total protein, totalnucleic acid and 16S rRNA, are presented in FIGS. 14(b)-14(d). Thoughthe amplitude has a deterministic effect on the release of theintracellular materials, the level of lysis as judged by total proteinand nucleic acid release is much lower than the case of the comparativemethod of GB lysis. However, in the case of the 16S rRNA assay, despiteless efficient total nucleic acid release, higher signal of 16S rRNA wasdetected for electrical lysis (with 190 V) compared to the GB lysis.This finding indicates the significance of the accessibility of thespecific hybridisation region on the rRNA in addition to the rRNAconcentration in the cell lysate. The capability of the electricalmethod to modify macromolecular conformation appears to provide moreeffective assay-ready cell lysate preparation for rRNA assays.

Example 2.2 Effects of Train Duration and Ionic Strength on ElectricalLysis Efficiency

The bacteria suspension of 1×10⁹ CFU/mL in 0.1 or 0.4 mM phosphatebuffer pH 7.4 was passed through a narrow channel in steps of 5 μL/10 sand 10 kHz and 155 V pulse trains with different durations were appliedto the suspension. The current envelopes are presented in FIG. 15(a).

The results of 3 analytical assays are presented in FIGS. 15(B)-15(D).In general, the release of intracellular materials increases drasticallywhen the train duration is close to the critical duration t_(c). In thecase of 0.1 mM ionic strength, though the pulse duration approachest_(c), the level of released molecules is well below the correspondingcase of higher ionic strength. This implies that the heating rate mayhave a deterministic effect on the electric lysis and treatment.

Example 2.3 The State of Proteins in the Electrically Prepared Lysate

In order to demonstrate the action of electrical treatment on thebiomolecules, the activity of the endogenous enzyme beta-galactosidase,expressed by the pUC19 plasmid vector, was measured. To detect theenzyme activity of beta-galactosidase, the cell lysate of the previousexample was centrifuged at 7000 rpm for 5 min and 50 μL of thesupernatant was mixed with an equal volume of 2-Nitrophenylβ-D-galactopyranoside (N-1127, Sigma) 4 mg/mL in 0.1 mM phosphate bufferpH 7.4. The color development was measured by spectrophotometer atabsorbance 420 nm. The measured residual enzyme activity, normalized tothe activity in the case of GB lysis, is presented in FIG. 15(E). Again,the increase in the train duration close to and beyond t_(c) have asignificant effect on the deactivation of the enzyme. This findingindicates that electrical lysis modifies the conformation ofmacromolecules.

Example 2.4 The State of Nucleic Acids in the Electrically PreparedLysate

To assess the spectrum of different types of nucleic acids released byelectrical lysis, in the example of section 2.2, the cell lysates werecentrifuged at 10000 rpm for 5 min and the nucleic acids in thesupernatants were resolved by gel electrophoresis on 0.5% agarose gel in0.5×TBE buffer and 0.5 μg/mL ethidium bromide (EtBr) at 150 volts for 45min. The release of genomic DNA, plasmid DNA and total RNA by electricallysis was shown in FIG. 15(F).

In comparison with GB lysis, the released genomic DNA by electricallysis was mainly observed as a slower mobility band with lessfluorescence intensity. To further clarify this observation, electricaltreatment was applied to the purified genomic DNA prepared by usingGenElute Bacterial Genomic DNA kit (NA2100, Sigma) and the results wereshown in FIG. 16.

DH5-alpha competent E. coli cells were grown on LB agar plates and asingle colony of E. coli was cultured in LB broth overnight at 37° C.Following the manufacturer's protocol, the cells were pre-treated withRNase A and Proteinase K solutions prior to the addition of cell lysissolution and incubation at 55° C. for 10 min. The genomic DNA, purifiedby using a spin column and eluted in nuclease-free water, wasre-suspended in 0.2 mM phosphate buffer pH 7.4 to an equivalentconcentration of 0.5×10⁹ CFU/mL.

Purified genomic DNA was passed through a wide and long channel in stepsof 10 μL/10 s and electrically treated with train durations of 34 or 44ms and pulse amplitude of 140 V. The current profile is presented inFIG. 16(a). Genomic DNA with or without electrical treatment wasresolved by agarose gel electrophoresis and the results are presented inFIG. 16(b). The electrical treatment resulted in a mobility shift ofgenomic DNA as was observed in FIG. 15 f, indicating the change inconformation of genomic DNA during electrical lysis.

The plasmid DNA released by electrical lysis also was resolved as aslower mobility band in FIG. 15(F). To further clarify this observation,electrical treatment was applied to the purified pUC19 plasmid DNAprepared by using GenElute Plasmid Miniprep kit (PLN10, Sigma)) and theresults were shown in FIG. 16.

NEB 5-alpha competent E. coli cells, transformed with pUC19 plasmid weregrown on LB agar plates supplemented with 100 μg/mL Ampicillin. A singleblue colony of E. coli was cultured in LB broth supplemented with 100μg/mL Ampicillin overnight at 37° C. Following the manufacturer'sprotocol, the cells were lysed in sodium hydroxide lysing buffer andre-suspended in the neutralization/binding buffer. The plasmid, purifiedby using a spin column and eluted in nuclease-free water, wasre-suspended in 0.2 mM phosphate buffer pH 7.4 to an equivalentconcentration of 1×10⁹ CFU/mL.

Purified pUC19 plasmid was passed through a wide and long channel insteps of 10 μL/10 s and electrically treated with train durations of 34or 44 ms and pulse amplitude of 140 V. The current profile is presentedin FIG. 16(a). Plasmid DNA with or without electrical treatment wasresolved by agarose gel electrophoresis and as indicated in FIG. 16(c)the electrical treatment results in a mobility shift similar to what wasobserved following the electrical lysis in FIG. 15(f), indicating thechange in conformation of plasmid DNA during electrical lysis.

Example 2.5 Capability of the Device to Prepare Sample for PolymeraseChain Reaction (PCR)

This example demonstrates the effectiveness of the electrical treatmentaction of the device in terms of reducing the inhibitory factors of PCR,enabling direct PCR processing without the need for additional reagentsor process. Bacteria-specific 16S rRNA gene (rDNA) was amplified by PCR,using Bacteria Identification Kit (BioChain Institute, Inc.). Theexperiments were performed by lysing NEB 5-alpha competent E. coli cellsre-suspended in 0.1 and 0.4 mM phosphate buffer pH 7.4.

Although total cell lysate can be used as an assay-ready sample, todemonstrate the efficient release of genomic DNA out of the cells, thesupernatant of the cell lysate was used for PCR. The cell lysateprepared by electrical lysis was centrifuged at 7000 rpm for 5 min andthe supernatant containing released genomic DNA was collected. As areference cell lysis control, cells were lysed mechanically by vortexingwith an equal volume of glass beads. PCR reaction was prepared by mixing1 μL of the cell lysate supernatant, 1 μL of universal control primermix, 12.5 μL of 2×PCR mix and 10.5 μL of TE buffer. Universal bacteriaspecific 16S rRNA gene fragment of 475 base pairs was amplified by 1cycle of 95° C. for 120 seconds, 35 cycles of 95° C. for 30 seconds, 56°C. for 45 seconds and 72° C. for 40 seconds, and 1 cycle of 72° C. for600 seconds. The resulting PCR product was resolved by gelelectrophoresis on 1.2% agarose gel in 0.5×TBE buffer and 0.5 μg/mLethidium bromide at 150 volts for 30 min.

A fragment of 16S rDNA was efficiently amplified from all electricallysates prepared by different electrical parameters as indicated in FIG.15(G), but only poor amplification was observed for glass bead lysate.

PCR inhibition effect by the inhibitors associated with cellularcomponents of bacteria is a well known mechanism and this effect isusually overcome by sample dilution or using PCR inhibitor removal kitprior to PCR amplification. When the glass bead lysate was seriallydiluted prior to PCR reactions as recommended by Bacteria IdentificationKit protocol, successful PCR amplification was observed in FIG. 16(d),indicating the presence of PCR inhibitory factors in GB lysate.Tofurther demonstrate the capability of PCR-ready sample processing byelectrical lysis, electrical treatments were applied to the supernatantof GB lysate with potential PCR inhibitory factors by passing the lysatethrough a wide and long channel and applying 140 V pulse and 10 kHztrains with duration of 34 or 44 ms, for which the corresponding currentprofile is presented in FIG. 16(a). As shown in FIG. 16(d), electricaltreatment eliminates PCR inhibition effect present in GB lysate.

Example 2.6 Capability of the Device to Release Intact Plasmid DNA forDownstream Applications

This example demonstrates the capability of the device to releaseplasmid DNA with preserved integrity for downstream applications.

NEB 5-alpha competent E. coli cells, transformed with pUC19 plasmidwhich contains ampicillin resistance gene and beta-galactosidase gene,were cultured in LB broth supplemented with 100 μg/mL Ampicillinovernight at 37° C. The cells were centrifuged at 7000 rpm for 5 min.The cell pellet was washed twice and re-suspended in 0.4 mM phosphatebuffer pH 7.4 at a concentration of 1×10⁹ CFU/mL.

As a reference plasmid purification method, pUC19 plasmid was purifiedusing GenElute Miniprep kit (Sigma). Following the manufacturer'sprotocol, the cells were lysed in sodium hydroxide lysing buffer andre-suspended in the neutralization/binding buffer. During cell lysis,double-stranded nucleic acids of both genomic DNA and plasmid DNA aredenatured by the alkaline pH. During neutralization step, althoughplasmid DNA can re-nature back to double-stranded structure, denaturedgenomic DNA precipitates and is removed by centrifugation. The plasmidin the supernatant was purified using a spin column and eluted innuclease-free water. In the case of glass bead or, electrical lysis, thesupernatant of the cell lysate was mixed with the neutralization/bindingbuffer of GenElute Miniprep kit and the purification was continued asthe reference plasmid purification method.

The plasmids purified from 3 different types of cell lysis were resolvedby agarose gel electrophoresis and the release of plasmid by glass beadbeating or electrical lysis was presented in FIG. 16 e. Modification ofgenomic DNA structure as a result of electrical lysis by the devicecould be advantageous for plasmid DNA purification without therequirement of hazardous chemical lysis and macromolecular denaturationby sodium hydroxide. For example, after electrical treatment of thelysate, the genomic DNA, having an altered conformational state (asevidenced in FIGS. 15(F), 16(B) and 16(E), could be separated from theplasmid DNA by a separation method. The separation method could includepurification in a spin column and/or separation of the altered genomicDNA from the plasmid DNA using a filter.

To assess the integrity of the plasmid DNA for its possible downstreamapplications, pUC19 plasmid purified from GB lysis or electrical lysisand the reference plasmid purified by GenElute kit were transformed intoDH5-alpha strain of competent E. coli cells, using TransformAid Bacteriatransformation kit (Fermentas). The transformants were culturedovernight on LB agar plates supplemented with 100 μg/mL Ampicillin, 60μg/mL X-gal and 0.1 mM isopropylthio-β-D-galactosidase (IPTG) at 37° C.

The growth of transformed E. coli cells was observed on LB medium withAmpicillin and X-Gal with similar transformation efficiency for theplasmids obtained from 3 different sample preparation methods, as can beseen in FIGS. 16(f)-(i). This indicates the release of intact cloningquality plasmids by E-lysis.

EXAMPLE 3 Electrical Lysis of Streptococcus pneumoniae

The experiments described in this example are intended to demonstratethe effects of the electrical parameters on the efficiency of the devicefor lysing S. pneumoniae cells. ATCC 6303 strain S. pneumoniae cellswere grown on Trypticase Soy agar with 5% sheep blood and a singlecolony of S. pneumoniae was cultured in Tryptic Soy Broth overnight at37° C. For the lysis experiment, the cells were centrifuged at 10000 rpmfor 5 min. The cell pellet was washed twice and re-suspended in 0.2 to0.4 mM phosphate buffer pH 7.4 at a concentration of 1×10⁹ CFU/mL.

Cell lysis efficiency was assessed by total protein assay andquantitative total nucleic acid assay. As a reference cell lysiscontrol, cells were lysed mechanically by beating with an equal volumeof glass beads (106 μm, Sigma) for 2 min and the supernatant of glassbead cell lysate was assayed after centrifugation.

Example 3.1 Effects of Pulse Amplitude and Train Duration and IonicStrength on Electrical Lysis Efficiency

The bacteria suspension of 1x10⁹ CFU/mL concentration in 0.2 and 0.4 mMphosphate buffer pH 7.4 was passed through a wide channel in steps of 10μL/10 s and 20 kHz pulse trains with different durations and amplitudeswere applied to the suspension. The duration and amplitudes of any twodifferent parameter sets, (t₁, V₁) and (t₂, V₂), were related byt₁/t₂=(V₂/V₁)² to ensure nearly equivalent electrical power delivery tothe channel for both cases. The experiments were repeated for fivedifferent values of amplitude. The current envelopes for two amplitudelimits are presented in FIG. 17(A). For both ionic strengths the traindurations are longer than corresponding t_(c).

The results of two analytical assays, detections of total protein andtotal nucleic acid, are presented in FIGS. 17(B) and 17(C). The lysisefficiency is improved by increasing the pulse amplitude and the heatingrate.

Example 3.2 The State of Nucleic Acids in the Electrically PreparedLysate

To assess the spectrum of different types of nucleic acids released byelectrical lysis, the cell lysates were centrifuged at 10000 rpm for 5min and the nucleic acids in the supernatants were resolved by gelelectrophoresis on 0.5% agarose gel in 0.5×TBE buffer and 0.5 μg/mLethidium bromide (EtBr) at 150 volts for 45 min. The release of genomicDNA and total RNA by electrical lysis was observed in FIG. 17(d). Incomparison with glass bead lysis, the released genomic DNA by electricallysis was mainly observed as a slower mobility band with lessfluorescence intensity. Similar to the case of the E. coli, thisobservation was attributed to conformational change of the genomic DNAto relaxed state.

Example 3.3 Capability of the Device to Prepare Sample for PolymeraseChain Reaction (Streptococcus pneumoniae)

This example demonstrates the effectiveness of the lysate treatmentaction of the device in terms of reducing the inhibitory factors of PCR,enabling direct PCR processing without the need for additional reagents.Bacteria-specific 16S rRNA gene (rDNA) was amplified by PCR, usingBacteria Identification Kit (BioChain Institute, Inc.). The experimentswere performed by lysing Streptococcus pneumoniae cells under theparameter set similar to (0.4 mM, 240V), the case whose correspondingcurrent is represented in FIG. 17(a).

Although total cell lysate can be used as an assay-ready sample, todemonstrate the efficient release of genomic DNA out of the cells, thesupernatant of the cell lysate was used for PCR. The cell lysate wascentrifuged at 7000 rpm for 5 min and the supernatant containingreleased genomic DNA was collected. The cell lysate supernatant of 1 μLvolume was used for PCR amplification. The resulting PCR product of 475base pair fragment of 16S rDNA was resolved by gel electrophoresis on1.2% agarose gel in 0.5×TBE buffer and 0.5 μg/mL ethidium bromide at 150volts for 30 min. A fragment of 16S rDNA was efficiently amplified fromthe supernatant of electrical lysate as indicated in FIG. 17(e).

Example 3.4 Dependence of the Device Lysis Performance on ElectrodeMaterial

This study shows the advantages of using SEOA as electrode material interms of preserving macromolecule integrity during electrical lysis. S.pneumoniae cells, suspended in 0.4 mM phosphate buffer pH 7.4, werelysed in geometrically similar wide channels; one channel having SEOA2electrodes and the other having copper electrodes. The pulse amplitudeand frequency were 200 V and 20 kHz, respectively. The current envelopecorresponding to these cases are presented in FIG. 18(A).

The performances of the devices were assessed by running Bradfordprotein assays and total nucleic acid assays, following the protocols ofexample 3.1. The results are presented in FIGS. 18(b) and 18(c). Whilethe two channels are similar in terms of releasing proteins, the totalnucleic acid signal is very low for the case of copper electrode,indicating possible degradation of the nucleic acid molecules duringelectrical lysis. To further verify this observation PCR was performedon samples lysed by the two devices and the result was presented in FIG.18 d.

EXAMPLE 4 Electrical Lysis of Saccharomyces cerevisiae

The experiments described in this example are intended to demonstratethe effects of the electrical parameters on the efficiency of the devicefor lysing S. cerevisiae fungal cells. The cells were grown onTrypticase Soy agar with 5% sheep blood and a single colony of S.cerevisiae was cultured in Tryptic Soy Broth overnight at 37° C. For thelysis experiment, the cells were centrifuged at 10000 rpm for 5 min. Thecell pellet was washed twice and re-suspended in 0.4 mM phosphate bufferpH 7.4 at a concentration of 2.5×10⁷ CFU/mL.

Cell lysis efficiency was assessed by total protein assay andquantitative total nucleic acid assay. As a reference cell lysiscontrol, cells were lysed mechanically by beating with an equal volumeof glass beads (710-1180 μm, G1152 Sigma) for 2 min and the supernatantof GB was assayed after centrifugation.

The cell suspension was passed through a wide channel in steps of 10μL/10 s and 20 kHz pulse trains with a duration of 29 ms and pulseamplitude of 190 V. The experiments were repeated under two conditions,open and restricted ports. In the later case, a restriction in themovement of the liquid at the inlet and outlet ports enabledsuperheating. The current envelopes for the two cases are presented inFIG. 19(A). The estimated average temperature of the superheated liquidin the restricted channel was approximately 160° C.

The results of two analytical assays, measurements of total releasedprotein and nucleic acid, are presented in FIGS. 19(B) AND 19(C). Thelysis efficiency is substantially improved by superheating.

EXAMPLE 5 Capability of the Device to Prepare Assay-Ready Sample forReverse Transcription (RT)-PCR of rRNA

This example demonstrates the assay readiness of the lysate prepared inthe device by subjecting the lysate to enzymatic transcription of asection of rRNA followed by PCR amplification of the resulting cDNA. Thetests were performed using three wide channels with differing heights;h=100 μm, h=200 μm, and h=400 μm. The ionic strengths of the cellsuspensions lysed in these channels were, respectively, 0.4, 0.8 and 1.6mM, thus ensuring nearly similar initial ionic currents in the channelsfor a given applied voltage. Moreover, the liquid was injected indiffering steps of 5 4/10s (for h=100 μm), 10 μL/10 s (for h=200 μm) and20 μL/10 s (for h=400 μm) into the three channels such that each cellexperienced the 10 kHz and 160 V pulse trains twice. The currentenvelopes for the three cases are presented in FIG. 20(A).

Bacteria-specific 16S rRNA was detected by reverse transcriptionpolymerase chain reaction (RT-PCR), using SuperScript III One-StepRT-PCR system with Platinum Taq DNA polymerase (Invitrogen, LifeTechnologies). NEB5-alpha E. coli cells of 10⁴ CFU/mL in 0.4 to 1.6 mMphosphate buffer pH 7.4 were lysed by E-lysis. As reference cell lysismethods, cells were lysed mechanically by beating with an equal volumeof glass beads or thermally by incubation at 95° C. for 5 min. As anegative RT-PCR control, RT-PCR grade water (Invitrogen, LifeTechnologies) was used instead of the sample. RT-PCR reaction of 25 μLvolume was prepared by mixing 1 μL of sample (either the lysate, denotedby T, or the supernatant of the lysate, denoted by S), 12.5 μl of2×Reaction mix, 0.5 μl of forward primer (16S rRNA forward, 100,Integrated DNA Technology), 0.5 μl of reverse primer (BU1.3R, 100), 0.5μl of SuperScript III RT/Platinum Taq Mix, 10 μl of RT-PCR grade water.16s rRNA forward primer (5′-AGAGTTTGATCCTGGCTAG-3′) (SEQ. ID. 6) is acommercially available primer, and BU1.3R (5′-TAAGGTTCTTCGCGTTGCTT-3′)(SEQ. ID. 7) is a bacteria specific universal primer designed bysequence alignment software (Bioedit, Ibis Biosciences, USA) and primerdesign software (Primer3, National Institutes of Health). The 16S rRNAgene fragment of 992 base pairs was amplified by one-step RT-PCR byreverse transcription at 55° C. for 10 min, inactivation of reversetranscriptase at 94° C. for 2 min, followed by 30 cycles of cDNAamplification at 94° C. for 15 sec, 55° C. for 30 sec, and 68° C. for 45sec, and final extension at 68° C. for 5 min.

The resulting PCR product was resolved by gel electrophoresis on 1%agarose gel in 0.5×TBE buffer and 0.5 μg/mL ethidium bromide at 150volts for 30 min. RT-PCR product of rRNA derived from E-lysis ofNEBS-alpha E. coli cells was observed in FIG. 20(B) (where “S” denotesPCR performed on the supernatant of the lysate, and “T” denotes PCRperformed on the total lysate). The result indicated that while tallerchannels (h=200 and 400 μm) perform similar to glass bead beating, thesignal corresponding to h=100 μm channel is much higher, making thedevice more suitable for sensitive detection of bacterial cells.

EXAMPLE 6 Control of Electrical Treatment and Electrical Lysis viaElectrical Feedback Example 6.1 Fabrication of Device with Open andClosed Channels

In this example, two devices were constructed for which the inlet andoutlet channels were accordingly dimensioned so as to yield channelswhich were fluidically unrestricted (open) or restricted. With referenceto FIG. 5(D), devices were constructed with polycarbonate top and bottomplates (230 and 231), surface enhanced oxidized aluminum electrodes onthe channel top and bottom surfaces (225 and 226) and a polyimide spacerin which the electrical channel (220) and inlet and outlet channels (221and 222) were formed. The electrical channels for both open andrestricted conditions were 15 mm long, 6.4 mm wide and 0.2 mm in height.The inlet and outlet channels were 0.1 mm in height, 10 mm in length and1.5 mm and 0.5 mm in width for the open and restricted channelsrespectively.

The channels were exposed to atmospheric pressure at the entrance andexit ports and subjected to an applied voltage in the form of a squarebipolar pulse train with 200 pulses, a constant amplitude of 195 V and afrequency of 10 kHz. The current flowing across the channel is plottedin FIG. 21 for 0.8 mM phosphate buffer solution.

For the open channel, the characteristic increase of the electricalcurrent to a maximum value followed by a rapid decrease due to the onsetof a liquid to gas phase transition is displayed in FIG. 21. Asdescribed in detail in above, it is believed that the maximum currentrepresents a minimum electrical impedance of the fluid and is indicativeof the temperature of the fluid. At this point the fluid is in asaturated liquid state in some regions of the channel combined with someregions of initial vapor formation and zones of fluid at coolertemperatures near the boundaries. The subsequent decrease of current inthe open channel appears to result from the expansion of the vaporregion and the consequent momentary expulsion of fluid from the channelthrough the inlet and outlet ports into tubes mounted at the ports. Thedisplaced liquid and vapour rapidly cool, and condensation of the vapouroccurs returning the liquid to the channel momentarily followingexpulsion.

The electrical channel with restricted inlet and outlet achieves ahigher electrical current, and hence a lower impedance, indicating ahigher liquid temperature as compared to the case of an open channelwith similar channel dimensions. This is due to the buildup of pressurein the channel afforded by the higher fluidic resistance of the inletand outlet channels which suppresses phase change and allowstemperatures in excess of those in the open channel to be achieved.

In this example for the restricted channel electrical pulses were haltedat 0.02 seconds which prevented the occurrence of extended phase changeand vapour formation which would have led to a decrease in theelectrical current as occurred for the open channel example. The ratioof the initial impedance to the minimum impedance in FIG. 21 are 3.16and 2.55 for the restricted and open channels respectively whichillustrates the higher temperature achieved in the restricted channel.

The finite differences analysis of transient heat transfer in the fluidfilled electrical channel was used to estimate channel temperatures bothduring and following the electrical pulse train. Fourier's law of heatconduction was solved numerically in conjunction with conservation ofenergy for a channel spatially discretized across the thickness and thewidth of the layered channel assembly and for which temperature isassumed to be uniform over the length of the channel.

The finite differences analysis described earlier was used to analyseJoule heating of the channel fluid using measured voltage and currentand assuming a linear thermal dependence of the conductance of the fluidwith an assumed value of 2.5% per degree Celcius. The calculated averagechannel temperatures are presented in FIG. 22 for the open andrestricted channel example described above over a period of 0.04 secondsfor which the voltage pulse train was halted at 0.02 seconds. Thecalculations indicate that a central region of the channel achievessuperheated temperatures of approximately 120° C. and 160° C. for theopen and restricted channels respectively. Peak average channeltemperatures reached are 106° C. and 140° C. respectively. In anotherelectrical channel embodiment, described further in an example below,the inlet and outlet channels are equipped with valves (223 and 224 inFIG. 5 which allow the electrical channel to be effectively closedduring the application of the voltage pulse train. The buildup ofpressure in the channel can in this case be greater than the case of therestricted channel of the above example, thus higher fluid temperaturescan be achieved before reaching the boiling threshold. Pressure buildupin the channel cannot for practical purposes be allowed to increasearbitrarily since, depending on the structure and materials of which thechannel is constructed, the device will possess a failure pressure atwhich point the channel will leak or fail catastrophically.

Active control of the voltage pulse train may be employed to limit themaximum pressure experienced by the channel. Active control may also beused to avoid vapour formation with its concomitant expulsion of fluidfrom the channel which may be undesirable in some applications such as,for example, integration into a fluidic cartridge.

Example 6.2 Temperature Dependence of Electrical Lysis Efficiency inElectrical Channels with Restricted Inlet and Outlet Channels

This example demonstrates the dependence of electrical lysis efficiencyon the temperature at the electrical channel. C. albicans cells weregrown on tryptic soy agar with 5% sheep blood. A single colony of C.albicans was cultured in tryptic soy broth overnight at 37° C. The cellswere centrifuged at 7000 rpm for 5 min. The cell pellet was washed twiceand re-suspended in 0.8 mM phosphate buffer pH 7.4, which waspre-filtered through a 0.2 μm filter. Four aliquots of C. albicans cellsin phosphate buffer were subjected to voltage pulse trains of amplitude195 V and frequency 10 kHz which were halted at predetermined voltagetrain durations. The electrical channel was as described above withrestricted inlet and outlet channels. The current response of thechannel is similar to that presented in FIG. 21 and the selected traindurations are indicated by arrows. The measured electrical response andthe estimated channel temperatures are presented in FIG. 23 for thedesignated conditions.

Two hundred fungal cells were added into 1000 μL of filter-sterilized0.8 mM phosphate buffer. For each condition 70 μL of the sample waselectrically lysed in steps of 10 μL/10 s. As a reference methodcomparison control, 100 μL aliquot of the same cell suspension wasmechanically lysed using glass beads. An equal volume of glass beads(710-1180 μm, Sigma) was added to the cell suspension and the mixturewas vortexed at maximum speed for 5 min. Then the cell lysatesupernatant was collected after centrifugation. This lysis method willbe known as GB lysis.

Reverse transcription-PCR assay was performed on 5 μL of each lysate,which is equivalent to detection at a single cell level using KAPA SYBRFAST One-Step qRT-PCR Universal kit (KAPA Biosystems). As a negativeRT-PCR control, pre-filtered 0.8 mM phosphate buffer pH 7.4, used forcell suspension was added instead of the sample. The reversetranscription PCR protocol used UFF4 forward primer;5′-AATTTCTGCCCTATCAACTTTCG-3′ (SEQ. ID 1) and UFR4 reverse primer,5′-CCCAAGGTTCAACTACGAGCTT-3′ (SEQ. ID 2). The fungal specific primersare designed by sequence alignment software (Bioedit, Ibis Biosciences,USA) and primer design software (Primer3, National Institutes ofHealth), and synthesized by Invitrogen, Life Technologies. Reversetranscription PCR reaction of 20 μL volume was prepared by mixing 5 μLof 2×KAPA SYBR FAST qPCR 2×mastermix, 0.4 μL 50×KAPA RT mix, 0.5 μl offorward primer (10 μM), 0.5 μl of reverse primer (10 μM) and 3.6 μl ofnuclease-free water. The 18S rRNA gene fragment of 343 base pairs at ahypervariable region of all fungal species (nucleotide 296 to 639 usingCandida albicans AB013586 as a reference) was amplified. One-step realtime reverse transcription PCR was performed by reverse transcription at55° C. for 5 min, inactivation of reverse transcription at 95° C. for 2min, followed by 30 cycles of cDNA amplification at 95° C. for 3 sec,59° C. for 3 sec, and 72° C. for 3 sec in Eco real time PCR system(Illumina).

The RT-PCR fluorescence signals versus cycle number plots are presentedin FIG. 24(A). The standard deviation, σ, of the signal over the first10 cycles, where the signal is predominately background noise, iscalculated for each curve and a threshold signal level is decided to be6σ. The cycle number where the recorded signal exceeds the thresholdlevel is defined as CT. The determined CT values are presented in FIG.24(B). As it is observed the lysis efficiency, inferred from the CTvalues, increases with peak liquid temperature, approaching the GB lysisefficiency.

It is expected that higher lysis and treatment efficiency can beobtained with higher peak temperatures which can only be achieved in thepresent channel by further restriction or closure of the inlet andoutlet ports together with feedback control which is described inExample 6.4 below. Lysis and treatment efficiency may also be increasedby increasing the residence time at elevated temperatures using feedbackcontrol of the voltage pulses which is described in Example 6.3 below.This approach also offers a way to overcome a potential limitation onthe power available from the constant voltage power supply by increasingefficiency at lower power levels.

Example 6.3 Dependence of Electrical Lysis Efficiency in an ElectricalChannel on the Pulse Density over an Extended Residence Time

This example demonstrates the method of increasing the electrical lysisefficiency by extending the residence time. The method may improveelectrical lysis and treatment effectiveness and can also beadvantageous when the power supply is not able to deliver high currentsand the system design constraints require operation at lower channeltemperatures. The method is based on rapidly increasing the channeltemperature to a desired value corresponding to a preselected impedancesetpoint. Thereafter the pulse density is decreased such that theaverage channel temperature is maintained within desired bounds.

The measured current responses corresponding to five conditions arepresented in FIGS. 25(A)-25E). In all cases voltage pulses with aconstant amplitude of 195V and frequency 10 kHz were applied. In thecase of the first four conditions the channel temperature is brought tothe estimated maximum value of 67° C. in approximately 8 ms by thevoltage pulse train and feedback control was activated when the currentreached 3.9 Amps. Thereafter impedance feedback control was activatedintroducing delays between pulses in response to the impedance errorsignal. Controller parameters were varied to create the conditions 1, 2,3 and 4 whose resulting current behavior is shown in FIGS. 25(A) to25(D), respectively.

It can be seen from these plots that the pulse density during thecontrol period differed somewhat between conditions as did the abilityof the controller to accurately hold impedance at a constant value. Inall these cases, significant vapour formation in the channel wasprevented.

For the uncontrolled case, whose electrical response is shown in FIG. 25(E), the maximum pulse density is maintained at its maximum value forthe whole train duration of 35 ms. After approximately 16 ms followingthe onset of the pulse train the vapour formation threshold (attemperatures similar to the restricted channel of FIG. 22) is reachedand the liquid experiences rapid expulsion due to the formation of avapour phase causing a rapid decrease in the current.

C. albicans cell suspension containing two hundred fungal cells in 1000μL of filter-sterilized 0.8 mM phosphate buffer was prepared followingthe steps described in example 1. Reverse transcription-PCR assay wasperformed on 5 μL of each lysate, which is equivalent to detection at asingle cell level, using KAPA SYBR FAST One-Step qRT-PCR Universal kit(KAPA Biosystems). As a negative reverse transcription-PCR control,pre-filtered 0.8 mM phosphate buffer pH 7.4, used for cell suspensionwas added instead of the sample. The reverse transcription PCR protocolused UFF4 forward primer; 5′- AATTTCTGCCCTATCAACTTTCG -3′ and UFR4reverse primer, 5′-CCCAAGGTTCAACTACGAGCTT-3′. The fungal specificprimers are designed by sequence alignment software (Bioedit, IbisBiosciences, USA) and primer design software (Primer3, NationalInstitutes of Health), and synthesized by Invitrogen, Life Technologies.Reverse transcription PCR reaction of 20 μL volume was prepared bymixing 5 μL of sample, 10 μl of 2×KAPA SYBR FAST qPCR 2×mastermix, 0.4μL 50×KAPA RT mix , 0.5 μl of forward primer (10 μM), 0.5 μl of reverseprimer (10 μM) and 3.6 μl of nuclease-free water. The 18S rRNA genefragment of 343 base pairs at a hypervariable region of all fungalspecies (nucleotide 296 to 639 using Candida albicans AB013586 as areference) was amplified. One-step real time reverse transcription PCRwas performed by reverse transcription at 55° C. for 5 min, inactivationof reverse transcription at 95° C. for 2 min, followed by 30 cycles ofcDNA amplification at 95° C. for 3 sec, 59° C. for 3 sec, and 72° C. for3 sec in Eco real time PCR system (Illumina).

The reverse transcription-PCR fluorescence signals versus cycle numberplots are presented in FIG. 26(A). The standard deviation, σ, of thesignal over the first 10 cycles, where the signal is predominatelybackground noise, is calculated for each curve and a threshold signallevel is decided to be 6σ. The cycle number where the recorded signalexceeds the threshold level is defined as CT. The determined CT valuesare presented in FIG. 26(B). As it is observed the lysis efficiency,inferred from the CT values, has increased with increasing the pulsedensity.

Example 6.4 Efficient Lysis of Fungal Cells in a Closed ElectricalChannel Employing Voltage Amplitude Regulation Scheme

This example demonstrates the method of increasing the electrical lysisefficiency by superheating the liquid without increasing the currentbeyond some preselected level. The method may improve electrical lysisand treatment effectiveness and is also advantageous in the case ofclosed electrical channels whenever the power supply is not able todeliver high currents.

In this example, the cells were subjected to mechanical lysis usingglass beads as a reference method and electrical lysis under 2conditions: open and closed channel ports. The cell suspension of 1×10³CFU/mL in 0.6 mM phosphate buffer pH 7.4 was passed through the channelin steps of 10 μL/15 s and the pulse train was applied when thesuspension was brought to rest. The electrical response is presented inFIGS. 27 (A) and 27(B) for the two electrical channels. The initialvoltage pulse train consisted of approximately 185 V, 10 kHz pulses forwhich impedance feedback control was introduced when the current reachedapproximately 5.5 Amps. The voltage amplitude was regulated by thecontroller to control the channel impedance seen in FIG. 27(C).

The impedance minimum value was limited by the controller to preventchannel leakage and channel fluid vaporization.

Fungal rRNA in the cell lysate samples (10³ CFU/mL) was detected byreverse transcription-PCR, targeting a hypervariable region in 18S rRNAof all fungal species. Reverse transcription-PCR reaction of 25 μLvolume was prepared by mixing 10 μL of the sample (nominal 10 cells persample), 12.5 μl of 2×PCR reaction mix (2G Robust HotStart, KAPABiosystems), 1.2 μL of reverse transcriptase (GoScript, Promega), 0.65μl of forward primer (UFF3, 10 μM) and 0.65 μl of reverse primer (UFR3,10 μM). As a negative reverse transcription-PCR control, pre-filtered0.6 mM phosphate buffer pH 7.4, used for cell suspension was addedinstead of the sample. UFF3 forward primer(5′-AACGAAAGTTAGGGGATCGAAG-3′) (SEQ. ID. 3) and UFR3 reverse primer(5′-CTTTAAGTTTCAGCCTTGCGA-3′) (SEQ. ID. 4) are fungal specific primersdesigned by sequence alignment software (Bioedit, Ibis Biosciences, USA)and primer design software (Primer3, National Institutes of Health), andsynthesized by Invitrogen, Life Technologies. The 18S rRNA gene fragmentof 167 base pairs at a hypervariable region of all fungi species(nucleotides 940 to 1107 using Candida albicans AB013586 as a reference)was amplified by one-step reverse transcription-PCR by reversetranscription at 55° C. for 5 min, inactivation of reverse transcriptaseand activation of hot start DNA polymerase at 95° C. for 3 min, followedby 35 cycles of cDNA amplification at 95° C. for 3 sec, 58° C. for 3sec, and 72° C. for 3 sec, and final extension at 72° C. for 1 min.

The resulting reverse transcription-PCR product of 15 μL was resolved bygel electrophoresis on 1% agarose gel in 0.5×TBE buffer and 0.5 μg/mLethidium bromide at 150 volts for 30 min. The amplified region of 18SrRNA derived from E-lysis and GB lysis of C. albicans cells was observedin FIG. 28 (A). This example demonstrates the performance of electricallysis with closed channel electrical channel which is superior toelectrical lysis with open channel electrical channel and GB lysis.

As a quantitative method of detection, the specific nucleotide sequenceswithin the amplified region were detected using the molecular beacon.reverse transcription-PCR product of 5 μL was mixed with 1 μL of thebuffer containing 20 mM Tris-HCl pH 8, 10 mM KCl and 7 mM MgCl₂ as wellas 1 μM of the molecular beacon6-FAM-5′-CCGAGCCGTAGTCTTAACCATAAACTATGCGCT-3′-DABCYL (SEQ. ID. 5)(nucleotides 977 to 997 using Candida albicans AB013586 as a reference)designed to detect all fungal pathogens. The mixture was heated at 95°C. for 30 sec to denature the amplicon and the molecular beacon,followed by cooling to room temperature, allowing hybridization of themolecular beacon to the target sequence. The resulting mixture of 6 μLwas transferred to a micro-well and the fluorescence intensity of thesamples and negative buffer controls at excitation wavelength of 492 nmand emission wavelength of 517 nm was measured using fluorescencemicroscopy (LumaScope, Etaluma).

The fluorescence signals detected by molecular beacon for thecorresponding amplicons resolved by gel electrophoresis in FIG. 28(A)are presented in FIG. 28(B). The quantitative detection also shows theperformance of electrical lysis in the electrical channel with closedchannels is superior to electrical lysis in the electrical channel withopen channels and GB lysis.

The specific embodiments described above have been shown by way ofexample, and it should be understood that these embodiments may besusceptible to various modifications and alternative forms. It should befurther understood that the claims are not intended to be limited to theparticular forms disclosed, but rather to cover all modifications,equivalents, and alternatives falling within the spirit and scope ofthis disclosure.

Therefore what is claimed is:
 1. A method of electrically processing aliquid within a microfluidic device to modify the activity of at leastone enzyme within the liquid; the microfluidic device including: anupper planar substrate; a lower planar substrate; and a side wall havinga thickness on a submillimeter scale, wherein said upper planarsubstrate, said lower planar substrate and said side wall define achannel; an upper electrode provided on an inner surface of said upperplanar substrate; and a lower electrode provided on an inner surface ofsaid lower planar substrate; the method including: flowing the liquidinto the channel, the liquid having an ionic strength between 0.1 mM and100 mM; applying bipolar voltage pulses between the upper electrode andthe lower electrode such that the liquid is heated with a heating rateof at least 250 degrees per second; wherein the voltage pulses areapplied such that the liquid is heated to an elevated temperaturesufficient to effect modification of the activity of the enzyme.
 2. Themethod according to claim 1 wherein the liquid comprises one or morenucleic acids, and wherein the enzyme is modified under the action ofthe bipolar voltage pulses without compromising the integrity of thenucleic acids.
 3. The method according to claim 1 wherein the liquid isheated to a temperature sufficient for denaturing the enzyme.
 4. Themethod according to claim 1 wherein the enzyme is a nuclease.
 5. Themethod according to claim 4 wherein the nuclease is RNAse.
 6. The methodaccording to claim 1 wherein said bipolar voltage pulses are providedsuch that an electric field between 2 kV/cm and 30 kV/cm is generatedacross the thickness of the channel.
 7. The method according to claim 6wherein the liquid comprises one or more cells, and wherein at least onecell is lysed under the application of the bipolar voltage pulses. 8.The method according to claim 7 wherein the enzyme is a nucleasereleased by the at least one cell.
 9. The method according to claim 8wherein the nuclease is modified under the action of the bipolar voltagepulses without compromising the integrity of nucleic acids released bythe at least one cell.
 10. The method according to claim 8 wherein thenuclease is RNAse.
 11. The method according to claim 8 furthercomprising amplifying a sequence of a nucleic acid released by the atleast one cell, wherein the amplification is performed in the absence ofa subsequent nucleic acid extraction or purification step.
 12. Themethod according to claim 1 wherein the voltage pulses are applied suchthat Joule heating of the liquid occurs with a rate of at least 2000degrees per second.
 13. The method according to claim 1 wherein theelevated temperature is between 80 degrees Celsius and 200 degreesCelsius.
 14. The method according to claim 1 wherein the elevatedtemperature greater than or equal to a boiling temperature of the liquidat atmospheric pressure.
 15. The method according to claim 1 furthercomprising monitoring a current flowing between the upper electrode andthe lower electrode, and employing the current as a feedback parameterfor controlling the temperature of the liquid.
 16. The method accordingto claim 15 wherein the liquid is heated to a phase transitiontemperature, the method further comprising identifying an initial peakin the current as the onset of the phase transition.
 17. The methodaccording to claim 15 further comprising applying the voltage pulses tomaintain the temperature for a prescribed time duration, based on thecurrent.
 18. The method according to claim 1 further comprisingmonitoring an impedance of the channel and employing the impedance as afeedback parameter for controlling the temperature of the liquid. 19.The method according to claim 1 wherein the channel is open duringapplication of the voltage pulses.
 20. The method according to claim 19wherein the diameter of a port in fluid communication with the channelis sufficiently restricted in size such that the liquid is superheatedduring application of the voltage pulses.
 21. The method according toclaim 1 wherein the microfluidic device comprises a first port in flowcommunication with a first side of the channel and a second port in flowcommunication with a second side of the channel, the method furthercomprising closing the first port and the second port during theapplication of the voltage pulses such that a pressure within thechannel increases while applying the voltage pulses.
 22. The methodaccording to claim 1 further comprising the step of regulating apressure of the liquid within the channel while applying the voltagepulses.
 23. The method according to claim 22 wherein the microfluidicdevice includes a passive pressure regulation device, and whereinregulating the pressure of the liquid includes passively regulating thepressure of the liquid.
 24. The method according to claim 22 wherein themicrofluidic device includes an expansion chamber equipped with apressure relief valve in fluid communication with the channel, andwherein regulating the pressure of the liquid includes limiting amaximum pressure of the liquid while applying the voltage pulses. 25.The method according to claim 22 wherein regulating the pressure of theliquid includes maintaining the pressure within the channel whilesuperheating the liquid.
 26. The method according to claim 1 furthercomprising processing the liquid to reduce the ionic strength of theliquid prior to flowing the liquid into the channel.
 27. The methodaccording to claim 26 further comprising flowing the liquid through amixed ion exchange resin prior flowing the liquid into the channel.